Charlotte's journal.rtf

{\rtf1\adeflang1025\ansi\ansicpg1252\uc1\adeff31507\deff0\stshfdbch31506\stshfloch31506\stshfhich31506\stshfbi31507\deflang1033\deflangfe1033\themelang1033\themelangfe0\themelangcs0{\fonttbl{\f0\fbidi \froman\fcharset0\fprq2{\*\panose 02020603050405020304}Times New Roman;}{\f1\fbidi \fswiss\fcharset0\fprq2{\*\panose 020b0604020202020204}Arial;}
{\f2\fbidi \fmodern\fcharset0\fprq1{\*\panose 02070309020205020404}Courier New;}{\f3\fbidi \froman\fcharset2\fprq2{\*\panose 05050102010706020507}Symbol;}{\f10\fbidi \fnil\fcharset2\fprq2{\*\panose 05000000000000000000}Wingdings;}
{\f34\fbidi \froman\fcharset1\fprq2{\*\panose 02040503050406030204}Cambria Math;}{\f37\fbidi \fswiss\fcharset0\fprq2{\*\panose 020f0502020204030204}Calibri;}{\flomajor\f31500\fbidi \froman\fcharset0\fprq2{\*\panose 02020603050405020304}Times New Roman;}
{\fdbmajor\f31501\fbidi \froman\fcharset0\fprq2{\*\panose 02020603050405020304}Times New Roman;}{\fhimajor\f31502\fbidi \fswiss\fcharset0\fprq2{\*\panose 020f0302020204030204}Calibri Light;}
{\fbimajor\f31503\fbidi \froman\fcharset0\fprq2{\*\panose 02020603050405020304}Times New Roman;}{\flominor\f31504\fbidi \froman\fcharset0\fprq2{\*\panose 02020603050405020304}Times New Roman;}
{\fdbminor\f31505\fbidi \froman\fcharset0\fprq2{\*\panose 02020603050405020304}Times New Roman;}{\fhiminor\f31506\fbidi \fswiss\fcharset0\fprq2{\*\panose 020f0502020204030204}Calibri;}
{\fbiminor\f31507\fbidi \froman\fcharset0\fprq2{\*\panose 02020603050405020304}Times New Roman;}{\f39\fbidi \froman\fcharset238\fprq2 Times New Roman CE;}{\f40\fbidi \froman\fcharset204\fprq2 Times New Roman Cyr;}
{\f42\fbidi \froman\fcharset161\fprq2 Times New Roman Greek;}{\f43\fbidi \froman\fcharset162\fprq2 Times New Roman Tur;}{\f44\fbidi \froman\fcharset177\fprq2 Times New Roman (Hebrew);}{\f45\fbidi \froman\fcharset178\fprq2 Times New Roman (Arabic);}
{\f46\fbidi \froman\fcharset186\fprq2 Times New Roman Baltic;}{\f47\fbidi \froman\fcharset163\fprq2 Times New Roman (Vietnamese);}{\f49\fbidi \fswiss\fcharset238\fprq2 Arial CE;}{\f50\fbidi \fswiss\fcharset204\fprq2 Arial Cyr;}
{\f52\fbidi \fswiss\fcharset161\fprq2 Arial Greek;}{\f53\fbidi \fswiss\fcharset162\fprq2 Arial Tur;}{\f54\fbidi \fswiss\fcharset177\fprq2 Arial (Hebrew);}{\f55\fbidi \fswiss\fcharset178\fprq2 Arial (Arabic);}
{\f56\fbidi \fswiss\fcharset186\fprq2 Arial Baltic;}{\f57\fbidi \fswiss\fcharset163\fprq2 Arial (Vietnamese);}{\f59\fbidi \fmodern\fcharset238\fprq1 Courier New CE;}{\f60\fbidi \fmodern\fcharset204\fprq1 Courier New Cyr;}
{\f62\fbidi \fmodern\fcharset161\fprq1 Courier New Greek;}{\f63\fbidi \fmodern\fcharset162\fprq1 Courier New Tur;}{\f64\fbidi \fmodern\fcharset177\fprq1 Courier New (Hebrew);}{\f65\fbidi \fmodern\fcharset178\fprq1 Courier New (Arabic);}
{\f66\fbidi \fmodern\fcharset186\fprq1 Courier New Baltic;}{\f67\fbidi \fmodern\fcharset163\fprq1 Courier New (Vietnamese);}{\f409\fbidi \fswiss\fcharset238\fprq2 Calibri CE;}{\f410\fbidi \fswiss\fcharset204\fprq2 Calibri Cyr;}
{\f412\fbidi \fswiss\fcharset161\fprq2 Calibri Greek;}{\f413\fbidi \fswiss\fcharset162\fprq2 Calibri Tur;}{\f416\fbidi \fswiss\fcharset186\fprq2 Calibri Baltic;}{\f417\fbidi \fswiss\fcharset163\fprq2 Calibri (Vietnamese);}
{\flomajor\f31508\fbidi \froman\fcharset238\fprq2 Times New Roman CE;}{\flomajor\f31509\fbidi \froman\fcharset204\fprq2 Times New Roman Cyr;}{\flomajor\f31511\fbidi \froman\fcharset161\fprq2 Times New Roman Greek;}
{\flomajor\f31512\fbidi \froman\fcharset162\fprq2 Times New Roman Tur;}{\flomajor\f31513\fbidi \froman\fcharset177\fprq2 Times New Roman (Hebrew);}{\flomajor\f31514\fbidi \froman\fcharset178\fprq2 Times New Roman (Arabic);}
{\flomajor\f31515\fbidi \froman\fcharset186\fprq2 Times New Roman Baltic;}{\flomajor\f31516\fbidi \froman\fcharset163\fprq2 Times New Roman (Vietnamese);}{\fdbmajor\f31518\fbidi \froman\fcharset238\fprq2 Times New Roman CE;}
{\fdbmajor\f31519\fbidi \froman\fcharset204\fprq2 Times New Roman Cyr;}{\fdbmajor\f31521\fbidi \froman\fcharset161\fprq2 Times New Roman Greek;}{\fdbmajor\f31522\fbidi \froman\fcharset162\fprq2 Times New Roman Tur;}
{\fdbmajor\f31523\fbidi \froman\fcharset177\fprq2 Times New Roman (Hebrew);}{\fdbmajor\f31524\fbidi \froman\fcharset178\fprq2 Times New Roman (Arabic);}{\fdbmajor\f31525\fbidi \froman\fcharset186\fprq2 Times New Roman Baltic;}
{\fdbmajor\f31526\fbidi \froman\fcharset163\fprq2 Times New Roman (Vietnamese);}{\fhimajor\f31528\fbidi \fswiss\fcharset238\fprq2 Calibri Light CE;}{\fhimajor\f31529\fbidi \fswiss\fcharset204\fprq2 Calibri Light Cyr;}
{\fhimajor\f31531\fbidi \fswiss\fcharset161\fprq2 Calibri Light Greek;}{\fhimajor\f31532\fbidi \fswiss\fcharset162\fprq2 Calibri Light Tur;}{\fhimajor\f31535\fbidi \fswiss\fcharset186\fprq2 Calibri Light Baltic;}
{\fhimajor\f31536\fbidi \fswiss\fcharset163\fprq2 Calibri Light (Vietnamese);}{\fbimajor\f31538\fbidi \froman\fcharset238\fprq2 Times New Roman CE;}{\fbimajor\f31539\fbidi \froman\fcharset204\fprq2 Times New Roman Cyr;}
{\fbimajor\f31541\fbidi \froman\fcharset161\fprq2 Times New Roman Greek;}{\fbimajor\f31542\fbidi \froman\fcharset162\fprq2 Times New Roman Tur;}{\fbimajor\f31543\fbidi \froman\fcharset177\fprq2 Times New Roman (Hebrew);}
{\fbimajor\f31544\fbidi \froman\fcharset178\fprq2 Times New Roman (Arabic);}{\fbimajor\f31545\fbidi \froman\fcharset186\fprq2 Times New Roman Baltic;}{\fbimajor\f31546\fbidi \froman\fcharset163\fprq2 Times New Roman (Vietnamese);}
{\flominor\f31548\fbidi \froman\fcharset238\fprq2 Times New Roman CE;}{\flominor\f31549\fbidi \froman\fcharset204\fprq2 Times New Roman Cyr;}{\flominor\f31551\fbidi \froman\fcharset161\fprq2 Times New Roman Greek;}
{\flominor\f31552\fbidi \froman\fcharset162\fprq2 Times New Roman Tur;}{\flominor\f31553\fbidi \froman\fcharset177\fprq2 Times New Roman (Hebrew);}{\flominor\f31554\fbidi \froman\fcharset178\fprq2 Times New Roman (Arabic);}
{\flominor\f31555\fbidi \froman\fcharset186\fprq2 Times New Roman Baltic;}{\flominor\f31556\fbidi \froman\fcharset163\fprq2 Times New Roman (Vietnamese);}{\fdbminor\f31558\fbidi \froman\fcharset238\fprq2 Times New Roman CE;}
{\fdbminor\f31559\fbidi \froman\fcharset204\fprq2 Times New Roman Cyr;}{\fdbminor\f31561\fbidi \froman\fcharset161\fprq2 Times New Roman Greek;}{\fdbminor\f31562\fbidi \froman\fcharset162\fprq2 Times New Roman Tur;}
{\fdbminor\f31563\fbidi \froman\fcharset177\fprq2 Times New Roman (Hebrew);}{\fdbminor\f31564\fbidi \froman\fcharset178\fprq2 Times New Roman (Arabic);}{\fdbminor\f31565\fbidi \froman\fcharset186\fprq2 Times New Roman Baltic;}
{\fdbminor\f31566\fbidi \froman\fcharset163\fprq2 Times New Roman (Vietnamese);}{\fhiminor\f31568\fbidi \fswiss\fcharset238\fprq2 Calibri CE;}{\fhiminor\f31569\fbidi \fswiss\fcharset204\fprq2 Calibri Cyr;}
{\fhiminor\f31571\fbidi \fswiss\fcharset161\fprq2 Calibri Greek;}{\fhiminor\f31572\fbidi \fswiss\fcharset162\fprq2 Calibri Tur;}{\fhiminor\f31575\fbidi \fswiss\fcharset186\fprq2 Calibri Baltic;}
{\fhiminor\f31576\fbidi \fswiss\fcharset163\fprq2 Calibri (Vietnamese);}{\fbiminor\f31578\fbidi \froman\fcharset238\fprq2 Times New Roman CE;}{\fbiminor\f31579\fbidi \froman\fcharset204\fprq2 Times New Roman Cyr;}
{\fbiminor\f31581\fbidi \froman\fcharset161\fprq2 Times New Roman Greek;}{\fbiminor\f31582\fbidi \froman\fcharset162\fprq2 Times New Roman Tur;}{\fbiminor\f31583\fbidi \froman\fcharset177\fprq2 Times New Roman (Hebrew);}
{\fbiminor\f31584\fbidi \froman\fcharset178\fprq2 Times New Roman (Arabic);}{\fbiminor\f31585\fbidi \froman\fcharset186\fprq2 Times New Roman Baltic;}{\fbiminor\f31586\fbidi \froman\fcharset163\fprq2 Times New Roman (Vietnamese);}}
{\colortbl;\red0\green0\blue0;\red0\green0\blue255;\red0\green255\blue255;\red0\green255\blue0;\red255\green0\blue255;\red255\green0\blue0;\red255\green255\blue0;\red255\green255\blue255;\red0\green0\blue128;\red0\green128\blue128;\red0\green128\blue0;
\red128\green0\blue128;\red128\green0\blue0;\red128\green128\blue0;\red128\green128\blue128;\red192\green192\blue192;}{\*\defchp \f31506\fs22 }{\*\defpap \ql \li0\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\adjustright\rin0\lin0\itap0 }\noqfpromote {\stylesheet{\ql \li0\ri0\sa160\sl259\slmult1\widctlpar\wrapdefault\aspalpha\aspnum\faauto\adjustright\rin0\lin0\itap0 \rtlch\fcs1 \af31507\afs22\alang1025 
\ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 \snext0 \sqformat \spriority0 Normal;}{\*\cs10 \additive \ssemihidden \sunhideused \spriority1 Default Paragraph Font;}{\*
\ts11\tsrowd\trftsWidthB3\trpaddl108\trpaddr108\trpaddfl3\trpaddft3\trpaddfb3\trpaddfr3\trcbpat1\trcfpat1\tblind0\tblindtype3\tsvertalt\tsbrdrt\tsbrdrl\tsbrdrb\tsbrdrr\tsbrdrdgl\tsbrdrdgr\tsbrdrh\tsbrdrv \ql \li0\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\adjustright\rin0\lin0\itap0 \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 \snext11 \ssemihidden \sunhideused Normal Table;}{
\s15\ql \li720\ri0\sa160\sl259\slmult1\widctlpar\wrapdefault\aspalpha\aspnum\faauto\adjustright\rin0\lin720\itap0\contextualspace \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 
\sbasedon0 \snext15 \sqformat \spriority34 \styrsid7687704 List Paragraph;}{\*\ts16\tsrowd\trbrdrt\brdrs\brdrw10 \trbrdrl\brdrs\brdrw10 \trbrdrb\brdrs\brdrw10 \trbrdrr\brdrs\brdrw10 \trbrdrh\brdrs\brdrw10 \trbrdrv\brdrs\brdrw10 
\trftsWidthB3\trpaddl108\trpaddr108\trpaddfl3\trpaddft3\trpaddfb3\trpaddfr3\trcbpat1\trcfpat1\tblind0\tblindtype3\tsvertalt\tsbrdrt\tsbrdrl\tsbrdrb\tsbrdrr\tsbrdrdgl\tsbrdrdgr\tsbrdrh\tsbrdrv 
\ql \li0\ri0\widctlpar\wrapdefault\aspalpha\aspnum\faauto\adjustright\rin0\lin0\itap0 \rtlch\fcs1 \af31507\afs24\alang1025 \ltrch\fcs0 \fs24\lang1033\langfe3082\loch\f31506\hich\af31506\dbch\af31505\cgrid\langnp1033\langfenp3082 
\sbasedon11 \snext16 \spriority59 \styrsid4467404 Table Grid;}}{\*\listtable{\list\listtemplateid1947118790\listhybrid{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\levelspace0\levelindent0{\leveltext
\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li720\lin720 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691
\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li1440\lin1440 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 
\fi-360\li2160\lin2160 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li2880\lin2880 }{\listlevel
\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li3600\lin3600 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0
\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li4320\lin4320 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1
\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li5040\lin5040 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0
\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li5760\lin5760 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext
\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li6480\lin6480 }{\listname ;}\listid198209100}{\list\listtemplateid-2089132808\listhybrid{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1
\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li720\lin720 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\levelspace0\levelindent0{\leveltext
\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li1440\lin1440 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}
\f10\fbias0 \fi-360\li2160\lin2160 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li2880\lin2880 }
{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li3600\lin3600 }{\listlevel\levelnfc23\levelnfcn23\leveljc0
\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li4320\lin4320 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0
\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li5040\lin5040 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative
\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li5760\lin5760 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext
\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li6480\lin6480 }{\listname ;}\listid300617883}{\list\listtemplateid1433183938\listhybrid{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\levelspace0
\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li360\lin360 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext
\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li1080\lin1080 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693
\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li1800\lin1800 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}
\f3\fbias0 \fi-360\li2520\lin2520 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li3240\lin3240 }
{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li3960\lin3960 }{\listlevel\levelnfc23\levelnfcn23
\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li4680\lin4680 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0
\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li5400\lin5400 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0
\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li6120\lin6120 }{\listname ;}\listid442580272}{\list\listtemplateid623278802\listhybrid{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0
\levelstartat1\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li720\lin720 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0
\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li1440\lin1440 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext
\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li2160\lin2160 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689
\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li2880\lin2880 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 
\fi-360\li3600\lin3600 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li4320\lin4320 }{\listlevel
\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li5040\lin5040 }{\listlevel\levelnfc23\levelnfcn23\leveljc0
\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li5760\lin5760 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1
\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li6480\lin6480 }{\listname ;}\listid676422088}{\list\listtemplateid164536266\listhybrid{\listlevel\levelnfc23\levelnfcn23\leveljc0
\leveljcn0\levelfollow0\levelstartat1\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li720\lin720 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\levelspace0
\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li1440\lin1440 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\levelspace0\levelindent0{\leveltext\leveltemplateid67698693
\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li2160\lin2160 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}
\f3\fbias0 \fi-360\li2880\lin2880 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li3600\lin3600 }
{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li4320\lin4320 }{\listlevel\levelnfc23\levelnfcn23
\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li5040\lin5040 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0
\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li5760\lin5760 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0
\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li6480\lin6480 }{\listname ;}\listid680008681}{\list\listtemplateid301125476\listhybrid{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0
\levelstartat1\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li720\lin720 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\levelspace0\levelindent0{\leveltext
\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li1440\lin1440 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}
\f10\fbias0 \fi-360\li2160\lin2160 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li2880\lin2880 }
{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li3600\lin3600 }{\listlevel\levelnfc23\levelnfcn23\leveljc0
\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li4320\lin4320 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0
\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li5040\lin5040 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative
\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li5760\lin5760 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext
\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li6480\lin6480 }{\listname ;}\listid811171715}{\list\listtemplateid-321345234\listhybrid{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\levelspace0
\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li720\lin720 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\levelspace0\levelindent0{\leveltext\leveltemplateid67698691
\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li1440\lin1440 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 
\fi-360\li2160\lin2160 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li2880\lin2880 }{\listlevel
\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li3600\lin3600 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0
\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li4320\lin4320 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1
\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li5040\lin5040 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0
\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li5760\lin5760 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext
\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li6480\lin6480 }{\listname ;}\listid846865052}{\list\listtemplateid582110114\listhybrid{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\levelspace0
\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li720\lin720 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\levelspace0\levelindent0{\leveltext\leveltemplateid67698691
\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li1440\lin1440 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 
\fi-360\li2160\lin2160 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li2880\lin2880 }{\listlevel
\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li3600\lin3600 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0
\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li4320\lin4320 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1
\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li5040\lin5040 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0
\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li5760\lin5760 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext
\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li6480\lin6480 }{\listname ;}\listid1019085904}{\list\listtemplateid-683655940\listhybrid{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1
\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li720\lin720 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\levelspace0\levelindent0{\leveltext
\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li1440\lin1440 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693
\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li2160\lin2160 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}
\f3\fbias0 \fi-360\li2880\lin2880 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li3600\lin3600 }
{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li4320\lin4320 }{\listlevel\levelnfc23\levelnfcn23
\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li5040\lin5040 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0
\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li5760\lin5760 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0
\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li6480\lin6480 }{\listname ;}\listid1059551229}{\list\listtemplateid146029438\listhybrid{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0
\levelstartat1\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li720\lin720 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\levelspace0\levelindent0{\leveltext
\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li1440\lin1440 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693
\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li2160\lin2160 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}
\f3\fbias0 \fi-360\li2880\lin2880 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li3600\lin3600 }
{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li4320\lin4320 }{\listlevel\levelnfc23\levelnfcn23
\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li5040\lin5040 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0
\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li5760\lin5760 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0
\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li6480\lin6480 }{\listname ;}\listid1146821656}{\list\listtemplateid-2079813062\listhybrid{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0
\levelstartat1\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li720\lin720 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\levelspace0\levelindent0{\leveltext
\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li1440\lin1440 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693
\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li2160\lin2160 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}
\f3\fbias0 \fi-360\li2880\lin2880 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li3600\lin3600 }
{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li4320\lin4320 }{\listlevel\levelnfc23\levelnfcn23
\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li5040\lin5040 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0
\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li5760\lin5760 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0
\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li6480\lin6480 }{\listname ;}\listid1149176536}{\list\listtemplateid1902269570\listhybrid{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0
\levelstartat1\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li720\lin720 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\levelspace0\levelindent0{\leveltext
\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li1440\lin1440 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693
\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li2160\lin2160 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}
\f3\fbias0 \fi-360\li2880\lin2880 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li3600\lin3600 }
{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li4320\lin4320 }{\listlevel\levelnfc23\levelnfcn23
\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li5040\lin5040 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0
\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li5760\lin5760 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0
\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li6480\lin6480 }{\listname ;}\listid1180312035}{\list\listtemplateid1725044480\listhybrid{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0
\levelstartat1\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li720\lin720 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\levelspace0\levelindent0{\leveltext
\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li1440\lin1440 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693
\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li2160\lin2160 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}
\f3\fbias0 \fi-360\li2880\lin2880 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li3600\lin3600 }
{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li4320\lin4320 }{\listlevel\levelnfc23\levelnfcn23
\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li5040\lin5040 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0
\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li5760\lin5760 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0
\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li6480\lin6480 }{\listname ;}\listid1186598126}{\list\listtemplateid652657936\listhybrid{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0
\levelstartat1\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li720\lin720 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\levelspace0\levelindent0{\leveltext
\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li1440\lin1440 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693
\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li2160\lin2160 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}
\f3\fbias0 \fi-360\li2880\lin2880 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li3600\lin3600 }
{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li4320\lin4320 }{\listlevel\levelnfc23\levelnfcn23
\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li5040\lin5040 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0
\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li5760\lin5760 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0
\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li6480\lin6480 }{\listname ;}\listid1233852701}{\list\listtemplateid-759815816\listhybrid{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0
\levelstartat1\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li720\lin720 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\levelspace0\levelindent0{\leveltext
\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li1440\lin1440 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}
\f10\fbias0 \fi-360\li2160\lin2160 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li2880\lin2880 }
{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li3600\lin3600 }{\listlevel\levelnfc23\levelnfcn23\leveljc0
\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li4320\lin4320 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0
\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li5040\lin5040 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative
\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li5760\lin5760 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext
\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li6480\lin6480 }{\listname ;}\listid1243416578}{\list\listtemplateid447228346\listhybrid{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\levelspace0
\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li720\lin720 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\levelspace0\levelindent0{\leveltext\leveltemplateid67698691
\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li1440\lin1440 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 
\fi-360\li2160\lin2160 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li2880\lin2880 }{\listlevel
\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li3600\lin3600 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0
\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li4320\lin4320 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1
\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li5040\lin5040 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0
\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li5760\lin5760 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext
\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li6480\lin6480 }{\listname ;}\listid1347321334}{\list\listtemplateid-879075724\listhybrid{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1
\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li720\lin720 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\levelspace0\levelindent0{\leveltext
\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li1440\lin1440 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693
\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li2160\lin2160 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}
\f3\fbias0 \fi-360\li2880\lin2880 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li3600\lin3600 }
{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li4320\lin4320 }{\listlevel\levelnfc23\levelnfcn23
\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li5040\lin5040 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0
\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li5760\lin5760 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0
\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li6480\lin6480 }{\listname ;}\listid1359232142}{\list\listtemplateid-274538794\listhybrid{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0
\levelstartat1\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li720\lin720 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0
\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li1440\lin1440 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext
\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li2160\lin2160 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689
\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li2880\lin2880 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 
\fi-360\li3600\lin3600 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li4320\lin4320 }{\listlevel
\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li5040\lin5040 }{\listlevel\levelnfc23\levelnfcn23\leveljc0
\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li5760\lin5760 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1
\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li6480\lin6480 }{\listname ;}\listid1520125425}{\list\listtemplateid779547264\listhybrid{\listlevel\levelnfc23\levelnfcn23\leveljc0
\leveljcn0\levelfollow0\levelstartat1\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li720\lin720 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\levelspace0
\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li1440\lin1440 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext
\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li2160\lin2160 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689
\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li2880\lin2880 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 
\fi-360\li3600\lin3600 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li4320\lin4320 }{\listlevel
\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li5040\lin5040 }{\listlevel\levelnfc23\levelnfcn23\leveljc0
\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li5760\lin5760 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1
\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li6480\lin6480 }{\listname ;}\listid1722514820}{\list\listtemplateid-595396026\listhybrid{\listlevel\levelnfc23\levelnfcn23
\leveljc0\leveljcn0\levelfollow0\levelstartat1\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li720\lin720 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1
\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li1440\lin1440 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext
\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li2160\lin2160 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689
\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li2880\lin2880 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 
\fi-360\li3600\lin3600 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li4320\lin4320 }{\listlevel
\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li5040\lin5040 }{\listlevel\levelnfc23\levelnfcn23\leveljc0
\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li5760\lin5760 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1
\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li6480\lin6480 }{\listname ;}\listid1759521176}{\list\listtemplateid2033615324\listhybrid{\listlevel\levelnfc23\levelnfcn23
\leveljc0\leveljcn0\levelfollow0\levelstartat1\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li720\lin720 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1
\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li1440\lin1440 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0
{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li2160\lin2160 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689
\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li2880\lin2880 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 
\fi-360\li3600\lin3600 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li4320\lin4320 }{\listlevel
\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li5040\lin5040 }{\listlevel\levelnfc23\levelnfcn23\leveljc0
\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li5760\lin5760 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1
\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li6480\lin6480 }{\listname ;}\listid1836651252}{\list\listtemplateid-1020214230\listhybrid{\listlevel\levelnfc23\levelnfcn23
\leveljc0\leveljcn0\levelfollow0\levelstartat1\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li720\lin720 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1
\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li1440\lin1440 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext
\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li2160\lin2160 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689
\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li2880\lin2880 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 
\fi-360\li3600\lin3600 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li4320\lin4320 }{\listlevel
\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li5040\lin5040 }{\listlevel\levelnfc23\levelnfcn23\leveljc0
\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li5760\lin5760 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1
\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li6480\lin6480 }{\listname ;}\listid1875729049}{\list\listtemplateid173546610\listhybrid{\listlevel\levelnfc23\levelnfcn23\leveljc0
\leveljcn0\levelfollow0\levelstartat1\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li720\lin720 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\levelspace0
\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li1440\lin1440 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext
\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li2160\lin2160 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689
\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li2880\lin2880 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 
\fi-360\li3600\lin3600 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li4320\lin4320 }{\listlevel
\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li5040\lin5040 }{\listlevel\levelnfc23\levelnfcn23\leveljc0
\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li5760\lin5760 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1
\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li6480\lin6480 }{\listname ;}\listid1882134068}{\list\listtemplateid260882982\listhybrid{\listlevel\levelnfc23\levelnfcn23\leveljc0
\leveljcn0\levelfollow0\levelstartat1\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li720\lin720 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\levelspace0
\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li1440\lin1440 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext
\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li2160\lin2160 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689
\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li2880\lin2880 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 
\fi-360\li3600\lin3600 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li4320\lin4320 }{\listlevel
\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li5040\lin5040 }{\listlevel\levelnfc23\levelnfcn23\leveljc0
\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li5760\lin5760 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1
\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li6480\lin6480 }{\listname ;}\listid2113276651}{\list\listtemplateid-153348298\listhybrid{\listlevel\levelnfc23\levelnfcn23
\leveljc0\leveljcn0\levelfollow0\levelstartat1\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li720\lin720 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1
\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li1440\lin1440 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0
{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li2160\lin2160 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689
\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li2880\lin2880 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 
\fi-360\li3600\lin3600 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li4320\lin4320 }{\listlevel
\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698689\'01\u-3913 ?;}{\levelnumbers;}\f3\fbias0 \fi-360\li5040\lin5040 }{\listlevel\levelnfc23\levelnfcn23\leveljc0
\leveljcn0\levelfollow0\levelstartat1\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698691\'01o;}{\levelnumbers;}\f2\fbias0 \fi-360\li5760\lin5760 }{\listlevel\levelnfc23\levelnfcn23\leveljc0\leveljcn0\levelfollow0\levelstartat1
\lvltentative\levelspace0\levelindent0{\leveltext\leveltemplateid67698693\'01\u-3929 ?;}{\levelnumbers;}\f10\fbias0 \fi-360\li6480\lin6480 }{\listname ;}\listid2122142604}}{\*\listoverridetable{\listoverride\listid811171715\listoverridecount0\ls1}
{\listoverride\listid676422088\listoverridecount0\ls2}{\listoverride\listid1347321334\listoverridecount0\ls3}{\listoverride\listid1186598126\listoverridecount0\ls4}{\listoverride\listid1836651252\listoverridecount0\ls5}{\listoverride\listid1149176536
\listoverridecount0\ls6}{\listoverride\listid1875729049\listoverridecount0\ls7}{\listoverride\listid2122142604\listoverridecount0\ls8}{\listoverride\listid680008681\listoverridecount0\ls9}{\listoverride\listid1722514820\listoverridecount0\ls10}
{\listoverride\listid198209100\listoverridecount0\ls11}{\listoverride\listid442580272\listoverridecount0\ls12}{\listoverride\listid1019085904\listoverridecount0\ls13}{\listoverride\listid1233852701\listoverridecount0\ls14}{\listoverride\listid1243416578
\listoverridecount0\ls15}{\listoverride\listid1882134068\listoverridecount0\ls16}{\listoverride\listid300617883\listoverridecount0\ls17}{\listoverride\listid846865052\listoverridecount0\ls18}{\listoverride\listid1180312035\listoverridecount0\ls19}
{\listoverride\listid1146821656\listoverridecount0\ls20}{\listoverride\listid2113276651\listoverridecount0\ls21}{\listoverride\listid1359232142\listoverridecount0\ls22}{\listoverride\listid1520125425\listoverridecount0\ls23}{\listoverride\listid1759521176
\listoverridecount0\ls24}{\listoverride\listid1059551229\listoverridecount0\ls25}}{\*\rsidtbl \rsid92495\rsid344921\rsid590140\rsid884068\rsid996618\rsid1193842\rsid2190281\rsid2430266\rsid2513443\rsid2718437\rsid2782476\rsid2784689\rsid2884371
\rsid3040168\rsid3873192\rsid4154470\rsid4467404\rsid5314342\rsid5340816\rsid5784579\rsid6060177\rsid6375002\rsid6761215\rsid7235815\rsid7687704\rsid8345116\rsid8921509\rsid9129488\rsid9591986\rsid9857289\rsid10373936\rsid10506450\rsid11091451
\rsid11340448\rsid13704892\rsid13771675\rsid14615753\rsid14826441\rsid15010177\rsid15151431\rsid15891047\rsid15993217}{\mmathPr\mmathFont34\mbrkBin0\mbrkBinSub0\msmallFrac0\mdispDef1\mlMargin0\mrMargin0\mdefJc1\mwrapIndent1440\mintLim0\mnaryLim1}{\info
{\author Charlotte}{\operator David Ayeke}{\creatim\yr2015\mo6\dy20\hr16\min15}{\revtim\yr2015\mo6\dy20\hr16\min15}{\version2}{\edmins0}{\nofpages1}{\nofwords2961}{\nofchars16881}{\nofcharsws19803}{\vern57439}}{\*\xmlnstbl {\xmlns1 http://schemas.microsof
t.com/office/word/2003/wordml}}\paperw12240\paperh15840\margl1440\margr1440\margt1440\margb1440\gutter0\ltrsect 
\widowctrl\ftnbj\aenddoc\trackmoves0\trackformatting1\donotembedsysfont1\relyonvml1\donotembedlingdata0\grfdocevents0\validatexml1\showplaceholdtext0\ignoremixedcontent0\saveinvalidxml0\showxmlerrors1\noxlattoyen
\expshrtn\noultrlspc\dntblnsbdb\nospaceforul\formshade\horzdoc\dgmargin\dghspace180\dgvspace180\dghorigin450\dgvorigin0\dghshow1\dgvshow1
\jexpand\viewkind5\viewscale100\pgbrdrhead\pgbrdrfoot\splytwnine\ftnlytwnine\htmautsp\nolnhtadjtbl\useltbaln\alntblind\lytcalctblwd\lyttblrtgr\lnbrkrule\nobrkwrptbl\snaptogridincell\allowfieldendsel\wrppunct
\asianbrkrule\rsidroot2782476\newtblstyruls\nogrowautofit\usenormstyforlist\noindnmbrts\felnbrelev\nocxsptable\indrlsweleven\noafcnsttbl\afelev\utinl\hwelev\spltpgpar\notcvasp\notbrkcnstfrctbl\notvatxbx\krnprsnet\cachedcolbal \nouicompat \fet0
{\*\wgrffmtfilter 2450}\nofeaturethrottle1\ilfomacatclnup0\ltrpar \sectd \ltrsect\linex0\endnhere\sectlinegrid360\sectdefaultcl\sftnbj {\*\pnseclvl1\pnucrm\pnstart1\pnindent720\pnhang {\pntxta .}}{\*\pnseclvl2\pnucltr\pnstart1\pnindent720\pnhang 
{\pntxta .}}{\*\pnseclvl3\pndec\pnstart1\pnindent720\pnhang {\pntxta .}}{\*\pnseclvl4\pnlcltr\pnstart1\pnindent720\pnhang {\pntxta )}}{\*\pnseclvl5\pndec\pnstart1\pnindent720\pnhang {\pntxtb (}{\pntxta )}}{\*\pnseclvl6\pnlcltr\pnstart1\pnindent720\pnhang 
{\pntxtb (}{\pntxta )}}{\*\pnseclvl7\pnlcrm\pnstart1\pnindent720\pnhang {\pntxtb (}{\pntxta )}}{\*\pnseclvl8\pnlcltr\pnstart1\pnindent720\pnhang {\pntxtb (}{\pntxta )}}{\*\pnseclvl9\pnlcrm\pnstart1\pnindent720\pnhang {\pntxtb (}{\pntxta )}}
\pard\plain \ltrpar\qc \li0\ri0\sa160\sl259\slmult1\widctlpar\wrapdefault\aspalpha\aspnum\faauto\adjustright\rin0\lin0\itap0\pararsid2782476 \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {
\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid2782476\charrsid5784579 Charlotte Bourg iGEM Journal}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid9591986\charrsid5784579 
\par }\pard \ltrpar\ql \li0\ri0\sa160\sl259\slmult1\widctlpar\wrapdefault\aspalpha\aspnum\faauto\adjustright\rin0\lin0\itap0\pararsid2782476 {\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid2782476\charrsid5784579 6/1/15
\par }{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid7687704\charrsid5784579 Fill in from journal}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid2782476\charrsid5784579 
\par }{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid7687704\charrsid5784579 6/2/15
\par Make 1.5 liters of Luria-Bertani (LB) agar and 1.5 liters of LB media}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid8345116\charrsid5784579  (in Zhang lab)}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid7687704\charrsid5784579 :
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af31507\afs22 \ltrch\fcs0 \f3\fs22\insrsid7687704\charrsid5784579 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard\plain \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls1\adjustright\rin0\lin720\itap0\pararsid7687704\contextualspace \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {\rtlch\fcs1 \af31507 
\ltrch\fcs0 \insrsid7687704\charrsid5784579 Recipe for 1 liter of total volume of LB:
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid7687704\charrsid5784579 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls1\ilvl1\adjustright\rin0\lin1440\itap0\pararsid7687704\contextualspace {\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid7687704\charrsid5784579 5}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid344921\charrsid5784579  }{
\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid7687704\charrsid5784579 g of yeast extract
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid7687704\charrsid5784579 \hich\af2\dbch\af31506\loch\f2 o\tab}10}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid344921\charrsid5784579  }{\rtlch\fcs1 \af31507 \ltrch\fcs0 
\insrsid7687704\charrsid5784579 g of NaCl
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid7687704\charrsid5784579 \hich\af2\dbch\af31506\loch\f2 o\tab}10}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid344921\charrsid5784579  }{\rtlch\fcs1 \af31507 \ltrch\fcs0 
\insrsid7687704\charrsid5784579 g of tryptone
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid7687704\charrsid5784579 \hich\af2\dbch\af31506\loch\f2 o\tab}15}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid344921\charrsid5784579  }{\rtlch\fcs1 \af31507 \ltrch\fcs0 
\insrsid7687704\charrsid5784579 g of agar (for LB agar only)
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af31507\afs22 \ltrch\fcs0 \f3\fs22\insrsid7687704\charrsid5784579 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls1\adjustright\rin0\lin720\itap0\pararsid7687704\contextualspace {\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid7687704\charrsid5784579 add 2}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid344921\charrsid5784579  }{
\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid7687704\charrsid5784579 L of water to a flask with 15}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid344921\charrsid5784579  }{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid7687704\charrsid5784579 g yeast extract, 30}{
\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid344921\charrsid5784579  }{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid7687704\charrsid5784579 g NaCl, and 30}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid344921\charrsid5784579  }{\rtlch\fcs1 \af31507 \ltrch\fcs0 
\insrsid7687704\charrsid5784579 g tryptone
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af31507\afs22 \ltrch\fcs0 \f3\fs22\insrsid7687704\charrsid5784579 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}stir until dissolved
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af31507\afs22 \ltrch\fcs0 \f3\fs22\insrsid7687704\charrsid5784579 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}add 500 mL of the dissolved solution to each of 3 bottles with 7.5}{\rtlch\fcs1 \af31507 \ltrch\fcs0 
\insrsid344921\charrsid5784579  }{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid7687704\charrsid5784579 mL of agar
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af31507\afs22 \ltrch\fcs0 \f3\fs22\insrsid7687704\charrsid5784579 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}divide the remaining solution between 2 1-L bottles
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af31507\afs22 \ltrch\fcs0 \f3\fs22\insrsid7687704\charrsid5784579 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}autoclave the bottles:
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid7687704\charrsid5784579 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls1\ilvl1\adjustright\rin0\lin1440\itap0\pararsid7687704\contextualspace {\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid7687704\charrsid5784579 loosen the lid of the bottles slightly
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid7687704\charrsid5784579 \hich\af2\dbch\af31506\loch\f2 o\tab}put autoclave tape crossing from the bottle onto the lid\emdash can move to the lid later
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid7687704\charrsid5784579 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls1\ilvl1\adjustright\rin0\lin1440\itap0\pararsid8345116\contextualspace {\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid7687704\charrsid5784579 
place sensor in a flask of water (would leave sensor in holder for hard goods)}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid8345116\charrsid5784579 
\par }\pard\plain \ltrpar\ql \li0\ri0\sa160\sl259\slmult1\widctlpar\wrapdefault\aspalpha\aspnum\faauto\adjustright\rin0\lin0\itap0\pararsid8345116 \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {
\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid8345116\charrsid5784579 Make overnight cultures from frozen stock to make electrocompetent cells the next day (in Moon lab):
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af31507\afs22 \ltrch\fcs0 \f3\fs22\insrsid8345116\charrsid5784579 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard\plain \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls2\adjustright\rin0\lin720\itap0\pararsid8345116\contextualspace \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {\rtlch\fcs1 \af31507 
\ltrch\fcs0 \insrsid8345116\charrsid5784579 strains: JM109 and MG1655
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af31507\afs22 \ltrch\fcs0 \f3\fs22\insrsid8345116\charrsid5784579 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}from glycerol stock, }{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid344921\charrsid5784579 
set up two 50mL cultures in 250-}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid8345116\charrsid5784579 mL flasks
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af31507\afs22 \ltrch\fcs0 \f3\fs22\insrsid8345116\charrsid5784579 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}grow }{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid8345116\charrsid5784579 
overnight}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid8345116\charrsid5784579 
\par }\pard\plain \ltrpar\ql \li0\ri0\sa160\sl259\slmult1\widctlpar\wrapdefault\aspalpha\aspnum\faauto\adjustright\rin0\lin0\itap0\pararsid8345116 \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {
\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid8345116\charrsid5784579 6/3/15
\par Electrocompetent prep of JM109 and MG1655 (in Moon lab):
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af31507\afs22 \ltrch\fcs0 \f3\fs22\insrsid8345116\charrsid5784579 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard\plain \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls3\adjustright\rin0\lin720\itap0\pararsid8345116\contextualspace \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {\rtlch\fcs1 \af31507 
\ltrch\fcs0 \insrsid8345116\charrsid5784579 add 25}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid344921\charrsid5784579  }{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid8345116\charrsid5784579 mL of each culture (JM109 and MG1655) into 500 mL of LB in a 2}{
\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid344921\charrsid5784579  }{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid8345116\charrsid5784579 L flask
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af31507\afs22 \ltrch\fcs0 \f3\fs22\insrsid8345116\charrsid5784579 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}grow at 37}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid8345116\charrsid5784579 
\'b0C and 250 rpm for ~1 hour}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid8345116\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid8345116\charrsid5784579 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls3\ilvl1\adjustright\rin0\lin1440\itap0\pararsid8345116\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid8345116\charrsid5784579 started at ~10:33}{\rtlch\fcs1 \af1 
\ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid344921\charrsid5784579  }{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid8345116\charrsid5784579 am, taken out at ~11:35}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid344921\charrsid5784579  }{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid8345116\charrsid5784579 am}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid8345116\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid8345116\charrsid5784579 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls3\adjustright\rin0\lin720\itap0\pararsid8345116\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid8345116\charrsid5784579 add a 10x dilution into cuvettes}{\rtlch\fcs1 
\af31507 \ltrch\fcs0 \insrsid8345116\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid8345116\charrsid5784579 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls3\ilvl1\adjustright\rin0\lin1440\itap0\pararsid8345116\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid8345116\charrsid5784579 900}{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid8345116\charrsid5784579  \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid8345116\charrsid5784579 L of H}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\sub\chshdng0\chcfpat0\chcbpat8\insrsid8345116\charrsid5784579 2}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid8345116\charrsid5784579 0 and 100 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid8345116\charrsid5784579 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid8345116\charrsid5784579 L of JM109}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid8345116\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid8345116\charrsid5784579 \hich\af2\dbch\af31506\loch\f2 o\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid8345116\charrsid5784579 900}{\rtlch\fcs1 
\ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid8345116\charrsid5784579  \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid8345116\charrsid5784579 L of H}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\sub\chshdng0\chcfpat0\chcbpat8\insrsid8345116\charrsid5784579 2}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid8345116\charrsid5784579 0 and 100 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid8345116\charrsid5784579 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid8345116\charrsid5784579 L of MG1655}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid8345116\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f10\fs22\insrsid8345116\charrsid5784579 \loch\af10\dbch\af31506\hich\f10 \'a7\tab}}\pard \ltrpar\s15\ql \fi-360\li2160\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls3\ilvl2\adjustright\rin0\lin2160\itap0\pararsid8345116\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid8345116\charrsid5784579 Mix each with the pipetter}{\rtlch\fcs1 
\af31507 \ltrch\fcs0 \insrsid8345116\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid8345116\charrsid5784579 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls3\ilvl1\adjustright\rin0\lin1440\itap0\pararsid8345116\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid8345116\charrsid5784579 blank cuvette: 1}{\rtlch\fcs1 \ab\af1 
\ltrch\fcs0 \cf1\chshdng0\chcfpat0\chcbpat8\insrsid344921\charrsid5784579  }{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid8345116\charrsid5784579 mL of water}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid8345116\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f10\fs22\insrsid8345116\charrsid5784579 \loch\af10\dbch\af31506\hich\f10 \'a7\tab}}\pard \ltrpar\s15\ql \fi-360\li2160\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls3\ilvl2\adjustright\rin0\lin2160\itap0\pararsid8345116\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid8345116\charrsid5784579 avoid touching the bottom area of the cuvettes}
{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid8345116\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid8345116\charrsid5784579 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls3\adjustright\rin0\lin720\itap0\pararsid8345116\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid8345116\charrsid5784579 take the OD}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\sub\chshdng0\chcfpat0\chcbpat8\insrsid8345116\charrsid5784579 600}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid8345116\charrsid5784579 :}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid8345116\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid8345116\charrsid5784579 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls3\ilvl1\adjustright\rin0\lin1440\itap0\pararsid8345116\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid8345116\charrsid5784579 open i-control application and use file \'93
cuvette 600\'94}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid8345116\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f10\fs22\insrsid8345116\charrsid5784579 \loch\af10\dbch\af31506\hich\f10 \'a7\tab}}\pard \ltrpar\s15\ql \fi-360\li2160\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls3\ilvl2\adjustright\rin0\lin2160\itap0\pararsid8345116\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid8345116\charrsid5784579 specific to Moon lab}{\rtlch\fcs1 \af31507 
\ltrch\fcs0 \insrsid8345116\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid5340816\charrsid5784579 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls3\ilvl1\adjustright\rin0\lin1440\itap0\pararsid5340816\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid5340816\charrsid5784579 click \'93apply blanking\'94
 and start blanking, then take measurements of other 2 cuvettes}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid5340816\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid8345116\charrsid5784579 \hich\af2\dbch\af31506\loch\f2 o\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid8345116\charrsid5784579 JM109 has OD .287}{
\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid5340816\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid8345116\charrsid5784579 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls3\ilvl1\adjustright\rin0\lin1440\itap0\pararsid8345116\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid8345116\charrsid5784579 MG1655 has OD .390}{\rtlch\fcs1 \af31507 
\ltrch\fcs0 \insrsid8345116\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid5340816\charrsid5784579 \hich\af2\dbch\af31506\loch\f2 o\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid5340816\charrsid5784579 
Desired range: 0.35 to 0.4}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid5340816\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f10\fs22\insrsid5340816\charrsid5784579 \loch\af10\dbch\af31506\hich\f10 \'a7\tab}}\pard \ltrpar\s15\ql \fi-360\li2160\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls3\ilvl2\adjustright\rin0\lin2160\itap0\pararsid5340816\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid5340816\charrsid5784579 
Put JM109 flask back in incubator for ~15 minutes starting at 11:56}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid344921\charrsid5784579  }{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid5340816\charrsid5784579 
am to try to increase OD}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid5340816\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f10\fs22\insrsid5340816\charrsid5784579 \loch\af10\dbch\af31506\hich\f10 \'a7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid5340816\charrsid5784579 
Leave MG1655 on bench top (growing more slowly)}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid5340816\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid5340816\charrsid5784579 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls3\ilvl1\adjustright\rin0\lin1440\itap0\pararsid5340816\contextualspace {\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid5340816\charrsid5784579 Second measurement of OD of JM109: 0.375
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af31507\afs22 \ltrch\fcs0 \f3\fs22\insrsid5340816\charrsid5784579 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls3\adjustright\rin0\lin720\itap0\pararsid5340816\contextualspace {\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid5340816\charrsid5784579 In cold (4}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid5340816\charrsid5784579 \'b0C) room, evenly distribute each culture into 2 centrifuge bottles (total of 4 bottles) and place in an ice bucket. Wait for 15-30 minutes.}{\rtlch\fcs1 \af31507 \ltrch\fcs0 
\insrsid5340816\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid5340816\charrsid5784579 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls3\ilvl1\adjustright\rin0\lin1440\itap0\pararsid5340816\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid5340816\charrsid5784579 Placed on ice at ~12:30}{\rtlch\fcs1 \af1 
\ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid344921\charrsid5784579  }{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid5340816\charrsid5784579 pm}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid5340816\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid5340816\charrsid5784579 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls3\adjustright\rin0\lin720\itap0\pararsid5340816\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid5340816\charrsid5784579 Keep on ice and bring bottles to centrifuge. Set at 4
\'b0C and 3000}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid344921\charrsid5784579  }{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid5340816\charrsid5784579 rpm for 25 minutes}{\rtlch\fcs1 \af31507 \ltrch\fcs0 
\insrsid5340816\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid5340816\charrsid5784579 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid5340816\charrsid5784579 
Return to cold room and discard supernatant by pouring quickly and gently }{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid5340816\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid5340816\charrsid5784579 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid5340816\charrsid5784579 
Suspend with total 500 mL of water (125}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid344921\charrsid5784579  }{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid5340816\charrsid5784579 
mL each bottle) and swirl until pellet disappears}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid5340816\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid5340816\charrsid5784579 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls3\adjustright\rin0\lin720\itap0\pararsid344921\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid5340816\charrsid5784579 Centrifuge at 4\'b0C}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid344921\charrsid5784579  and 3000 rpm for 25 minutes}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid344921\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid344921\charrsid5784579 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid344921\charrsid5784579 
In cold room, discard liquid and suspend pellet gently (by swirling) with total 220 mL of 10% glycerol (55 mL each bottle) until you do not see pellet}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid344921\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid344921\charrsid5784579 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid344921\charrsid5784579 
Aliquot contents of each bottle into 4 15-mL conical tubes (16 tubes total)}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid344921\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid344921\charrsid5784579 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid344921\charrsid5784579 Centrifuge at 4
\'b0C and 3000 rpm for 25 minutes}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid344921\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid344921\charrsid5784579 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid344921\charrsid5784579 In cold room, po
ur off the liquid and pipette out the remaining liquid}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid344921\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid344921\charrsid5784579 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid344921\charrsid5784579 
Resuspend pellets in 1 mL glycerol in each tube, then combine into 1 tube per strain}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid344921\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid344921\charrsid5784579 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid344921\charrsid5784579 Centrifuge at 4
\'b0C and 3000 rpm for 25 minutes}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid344921\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid344921\charrsid5784579 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid344921\charrsid5784579 
In cold room pour out liquid and pipette of remaining liquid}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid344921\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid344921\charrsid5784579 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid344921\charrsid5784579 
Add 1.5 mL of 10% glycerol to each tube and resuspend}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid344921\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid344921\charrsid5784579 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid344921\charrsid5784579 Aliquot 40 }{
\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid344921\charrsid5784579 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid344921\charrsid5784579 
L each into small labeled vials and place in a labeled box in the -80\'b0C freezer}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid344921\charrsid5784579 
\par }\pard\plain \ltrpar\ql \li0\ri0\sa160\sl259\slmult1\widctlpar\wrapdefault\aspalpha\aspnum\faauto\adjustright\rin0\lin0\itap0\pararsid344921 \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {
\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid344921\charrsid5784579 Transformation (in Moon lab):
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af31507\afs22 \ltrch\fcs0 \f3\fs22\insrsid344921\charrsid5784579 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard\plain \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls4\adjustright\rin0\lin720\itap0\pararsid344921\contextualspace \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {\rtlch\fcs1 \af31507 
\ltrch\fcs0 \insrsid344921\charrsid5784579 Take MG1655 and WM1788 electrocompetent cells out of -80}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid344921\charrsid5784579 \'b0C and place on ice}{\rtlch\fcs1 \af31507 \ltrch\fcs0 
\insrsid344921\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid344921\charrsid5784579 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid344921\charrsid5784579 Take
 PSL2397 (plasmid) out of -80\'b0C and place on ice}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid344921\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid344921\charrsid5784579 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid344921\charrsid5784579 Add 2 }{
\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid344921\charrsid5784579 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid344921\charrsid5784579 L of PSL2397 directly into MG1655 tube}{\rtlch\fcs1 \af31507 
\ltrch\fcs0 \insrsid344921\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid344921\charrsid5784579 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls4\ilvl1\adjustright\rin0\lin1440\itap0\pararsid344921\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid344921\charrsid5784579 
Just add; do not draw back up and down with pipette}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid344921\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid7235815\charrsid5784579 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls4\adjustright\rin0\lin720\itap0\pararsid7235815\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid7235815\charrsid5784579 Set pipette over 40 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid7235815\charrsid5784579 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid7235815\charrsid5784579 L and draw up the MG1655 and PSL2397 mixture and place into electroporation cuvette }{
\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid7235815\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid7235815\charrsid5784579 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid7235815\charrsid5784579 Tap
 cuvette to ensure cells are at bottom of cuvette\emdash should check to see that there are no gaps\emdash and place into electroporator; turn on}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid7235815\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid7235815\charrsid5784579 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid7235815\charrsid5784579 
Immediately add 500 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid7235815\charrsid5784579 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid7235815\charrsid5784579 
L of LB into the cuvette then pour into culture tube}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid7235815\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid7235815\charrsid5784579 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid7235815\charrsid5784579 
Place culture tube into incubator for 1 hour }{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid7235815\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid7235815\charrsid5784579 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls4\ilvl1\adjustright\rin0\lin1440\itap0\pararsid7235815\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid7235815\charrsid5784579 MG16
55 placed into incubator at 4:58 pm and had time constant of 5.5 ms}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid7235815\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid7235815\charrsid5784579 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls4\adjustright\rin0\lin720\itap0\pararsid7235815\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid7235815\charrsid5784579 Repeat with WM1788}{\rtlch\fcs1 \af31507 \ltrch\fcs0 
\insrsid7235815\charrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid7235815\charrsid5784579 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls4\ilvl1\adjustright\rin0\lin1440\itap0\pararsid7235815\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid7235815\charrsid5784579 
Placed into incubator at 5:04 pm and had time constant of 5.3 ms}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid7235815\charrsid5784579 
\par }\pard\plain \ltrpar\ql \li0\ri0\sa160\sl259\slmult1\widctlpar\wrapdefault\aspalpha\aspnum\faauto\adjustright\rin0\lin0\itap0\pararsid7235815 \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {
\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid344921\charrsid5784579 
\par }{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid7235815\charrsid5784579 6/4/15
\par }\pard \ltrpar\ql \li0\ri0\sa160\sl259\slmult1\widctlpar\wrapdefault\aspalpha\aspnum\faauto\adjustright\rin0\lin0\itap0 {\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid9129488 Isolate plasmid (Zhang lab):
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af31507\afs22 \ltrch\fcs0 \f3\fs22\insrsid9129488 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard\plain \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls7\adjustright\rin0\lin720\itap0\pararsid9129488\contextualspace \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {\rtlch\fcs1 \af31507 
\ltrch\fcs0 \insrsid9129488 Work within 30 cm of flame and flame lip of glass flask at opening and closing
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af31507\afs22 \ltrch\fcs0 \f3\fs22\insrsid9129488 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Add 50 mL of LB to conical 50 ml tube
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af31507\afs22 \ltrch\fcs0 \f3\fs22\insrsid9129488 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Add 50 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid9129488\charrsid5784579 \'ec}{
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid9129488\charrsid5784579 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid9129488  of chloramphenicol and mix }{\rtlch\fcs1 \af31507 \ltrch\fcs0 
\insrsid9129488\charrsid9129488 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid9129488 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid9129488 Pour into 125 mL flask}{\rtlch\fcs1 \af31507 
\ltrch\fcs0 \insrsid9129488\charrsid9129488 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid9129488 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid9129488 
Add 1.5 mL of PA2C-TesA (cells with plasmid to be isolated) with a 1000 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid9129488\charrsid5784579 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid9129488\charrsid5784579 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid9129488  pipette}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid9129488\charrsid9129488 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid9129488 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid9129488 
Place flask on shaker in warm room at 250 rpm (200 to 250 rpm in standard for E. coli)}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid9129488\charrsid9129488 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid9129488 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls7\ilvl1\adjustright\rin0\lin1440\itap0\pararsid9129488\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid9129488 Placed on shaker at 9:20 am \endash 
 leave there until ~ 2 or 3 pm}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid9129488\charrsid9129488 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid9129488 \hich\af2\dbch\af31506\loch\f2 o\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid9129488 Took out of warm room/off shaker at 2:12pm}{
\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid9129488\charrsid9129488 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid9129488 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls7\adjustright\rin0\lin720\itap0\pararsid9129488\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid9129488 Pour into a 50 mL conical tube}{\rtlch\fcs1 \af31507 \ltrch\fcs0 
\insrsid9129488\charrsid9129488 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid9129488 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid9129488 
Centrifuge until supernatant is clear and then pour off supernatant}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid9129488\charrsid9129488 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid9129488 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid9129488 Add 2.5 mL of resuspension bu
ffer and pipette in and out to resuspend until no pellet remains}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid9129488\charrsid9129488 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid9129488 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid9129488 
Add 2.75 mL of lysis buffer and invert a few times to mix}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid9129488\charrsid9129488 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid9129488 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls7\ilvl1\adjustright\rin0\lin1440\itap0\pararsid9129488\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid9129488 Solution is now viscous}{\rtlch\fcs1 \af31507 \ltrch\fcs0 
\insrsid9129488\charrsid9129488 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid9129488 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls7\adjustright\rin0\lin720\itap0\pararsid9129488\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid9129488 Add 4 mL of neutralization buffer and invert a few times to mix}{
\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid9129488\charrsid9129488 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid9129488 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls7\ilvl1\adjustright\rin0\lin1440\itap0\pararsid9129488\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid9129488 Solution is cloudy}{\rtlch\fcs1 \af31507 \ltrch\fcs0 
\insrsid9129488\charrsid9129488 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid9129488 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls7\adjustright\rin0\lin720\itap0\pararsid9129488\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid9129488 Centrifuge at 4700 rpm for 15 minutes}{\rtlch\fcs1 \af31507 
\ltrch\fcs0 \insrsid9129488\charrsid9129488 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid9129488 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls7\ilvl1\adjustright\rin0\lin1440\itap0\pararsid9129488\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid9129488 
Supernatant not completely clear after centrifuging, but that is fine because it will be filtered}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid9129488\charrsid9129488 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid9129488 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls7\adjustright\rin0\lin720\itap0\pararsid9129488\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid9129488 
Filter into a new conical tube using a syringe with a cotton ball in it}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid9129488\charrsid9129488 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid9129488 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls7\ilvl1\adjustright\rin0\lin1440\itap0\pararsid9129488\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid9129488 Filtered liquid is clear}{\rtlch\fcs1 \af31507 \ltrch\fcs0 
\insrsid9129488\charrsid9129488 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af31507\afs22 \ltrch\fcs0 \f3\fs22\insrsid9129488 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls7\adjustright\rin0\lin720\itap0\pararsid9129488\contextualspace {\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid9129488 Add 2.5 times the volume of the liquid of cold pure ethanol (~10 mL }{\rtlch\fcs1 \af31507 
\ltrch\fcs0 \insrsid9129488 {\field{\*\fldinst SYMBOL 224 \\f "Wingdings" \\s 11}{\fldrslt\f10\fs22}}}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid9129488  ~35 mL total volume
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af31507\afs22 \ltrch\fcs0 \f3\fs22\insrsid9129488 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Put in -20}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid9129488\charrsid5784579 \'b0C}{
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid9129488  for 20 minutes}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid9129488\charrsid9129488 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid9129488 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls7\ilvl1\adjustright\rin0\lin1440\itap0\pararsid9129488\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid9129488 Put in at ~4:00 pm}{\rtlch\fcs1 \af31507 \ltrch\fcs0 
\insrsid9129488\charrsid9129488 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid9129488 \hich\af2\dbch\af31506\loch\f2 o\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid9129488 Taken out at ~4:22 pm}{\rtlch\fcs1 \af31507 
\ltrch\fcs0 \insrsid9129488\charrsid9129488 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid9129488 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls7\adjustright\rin0\lin720\itap0\pararsid9129488\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid9129488 Centrifuge for 25 minutes at 4}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid9129488\charrsid5784579 \'b0C}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid9129488  at 4700 rpm, then check for a pellet}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid9129488\charrsid9129488 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid15993217 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid15993217 Pour off supernatant}{\rtlch\fcs1 \af31507 
\ltrch\fcs0 \insrsid9129488\charrsid15993217 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid15993217 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls7\ilvl1\adjustright\rin0\lin1440\itap0\pararsid15993217\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid15993217 Can tap the top of the tube against a paper towel to 
remove more ethanol}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid15993217\charrsid15993217 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid15993217 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls7\adjustright\rin0\lin720\itap0\pararsid15993217\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid15993217 
Add at least 20 mL of 70% EtOH and shake to break up the pellet, then fortex}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid15993217\charrsid15993217 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid15993217 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid15993217 Centrifuge at 3000 rpm for 7 minutes at 4}{
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid15993217\charrsid5784579 \'b0C}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid15993217\charrsid15993217 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid15993217 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid15993217 Pour off the supernatant}{\rtlch\fcs1 
\af31507 \ltrch\fcs0 \insrsid15993217\charrsid15993217 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid15993217 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid15993217 Resuspend in 500 }{\rtlch\fcs1 \ab\af1 
\ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid15993217\charrsid5784579 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid15993217\charrsid5784579 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid15993217 
 of TE RNAse A (20 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid15993217\charrsid5784579 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid15993217 g/mL)}{\rtlch\fcs1 \af31507 \ltrch\fcs0 
\insrsid15993217\charrsid15993217 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid15993217 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid15993217 
Add 5 times that volume (2.5 mL) of BNL buffer (from old miniprep kit) and pipette in and out to mix}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid15993217\charrsid15993217 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid15993217 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid15993217 Add 750 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid15993217\charrsid5784579 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid15993217\charrsid5784579 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid15993217 
 of the solution to a spin mini column and collection tube}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid15993217\charrsid15993217 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid15993217 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid15993217 
Balance centrifuge and spin for 1 minute at 12500 rpm; discard flow-through}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid15993217\charrsid15993217 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af31507\afs22 \ltrch\fcs0 \f3\fs22\insrsid15993217 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid15993217 Repeat with additional 750 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid15993217\charrsid5784579 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid15993217\charrsid5784579 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid15993217 
 until all of the solution has run through the column (centrifuge each time on the same settings and in the same column)}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid15993217\charrsid15993217 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid15993217 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid15993217 
Spin down for 2 minutes at 12500 rpm to get rid of the rest of ethanol}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid15993217\charrsid15993217 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid15993217 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls7\ilvl1\adjustright\rin0\lin1440\itap0\pararsid15993217\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid15993217 Optional step that was not performed: can put in 50}{
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid15993217\charrsid5784579 \'b0C}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid15993217  room for a few minutes to evaporate the rest of the ethanol}{\rtlch\fcs1 \af31507 
\ltrch\fcs0 \insrsid15993217\charrsid15993217 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid15993217 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls7\adjustright\rin0\lin720\itap0\pararsid15993217\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid15993217 Add 750 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid15993217\charrsid5784579 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid15993217\charrsid5784579 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid4154470 
 of wash buffer; spin and discard the flow-through}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid15993217\charrsid4154470 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid4154470 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid4154470 Repeat with additional 750 }{\rtlch\fcs1 
\ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid4154470\charrsid5784579 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid4154470\charrsid5784579 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid4154470  of wash buffer}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid4154470\charrsid4154470 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid4154470 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid4154470 
Place column into a new Eppendorf tube and place 40 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid4154470\charrsid5784579 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid4154470\charrsid5784579 L}{
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid4154470  off eluent (in this case, water) directly onto the bottom of the column without touching it with the pipette tip}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid4154470\charrsid4154470 

\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid4154470 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid4154470 Wait ~3 minutes}{\rtlch\fcs1 \af31507 
\ltrch\fcs0 \insrsid4154470\charrsid4154470 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid4154470 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid4154470 
Centrifuge at 13000 rpm for 2 minutes and label flow-through vial}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid4154470\charrsid4154470 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid4154470 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid4154470 
Take measurement on nanodrop and label the vial with the concentration}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid4154470 
\par }\pard\plain \ltrpar\ql \li0\ri0\sa160\sl259\slmult1\widctlpar\wrapdefault\aspalpha\aspnum\faauto\adjustright\rin0\lin0\itap0 \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {\rtlch\fcs1 
\af31507 \ltrch\fcs0 \insrsid5784579 Use SnapGene to design primers for the 14 sequences to be overexpressed:
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af31507\afs22 \ltrch\fcs0 \f3\fs22\insrsid5784579 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard\plain \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls5\adjustright\rin0\lin720\itap0\pararsid5784579\contextualspace \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {\rtlch\fcs1 \af31507 
\ltrch\fcs0 \insrsid5784579 Check for EcoRI and XhoI restriction sit}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid10373936 es within each of the sequences}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid5784579 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af31507\afs22 \ltrch\fcs0 \f3\fs22\insrsid10373936 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls5\adjustright\rin0\lin720\itap0\pararsid4467404\contextualspace {\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid10373936 Check for directionality on the plasmid: direct or complementary}{\rtlch\fcs1 \af31507 
\ltrch\fcs0 \insrsid4467404 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af31507\afs22 \ltrch\fcs0 \f3\fs22\insrsid10373936 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls5\adjustright\rin0\lin720\itap0\pararsid5784579\contextualspace {\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid10373936 If a restriction enzyme does not have sites within the 
sequence, add a site for that restriction enzyme to the appropriate primer and add 6 adenines beyond the site on the primer so that the restriction enzyme will work properly
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af31507\afs22 \ltrch\fcs0 \f3\fs22\insrsid10373936 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}If the restriction enzyme does have a site within the sequence, end the primer at 
the end of the sequence to be amplified to leave the ends blunt
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af31507\afs22 \ltrch\fcs0 \f3\fs22\insrsid10373936 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}If direct, add EcoRI to the 5\rquote  end and XhoI to the 3\rquote  end as appropriate
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af31507\afs22 \ltrch\fcs0 \f3\fs22\insrsid10373936 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}If complementary, add XhoI to the 5\rquote  end and EcoRI to the 3\rquote  end as appropriate
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af31507\afs22 \ltrch\fcs0 \f3\fs22\insrsid10373936 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls5\adjustright\rin0\lin720\itap0\pararsid6375002\contextualspace {\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid10373936 Maintain a T}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \sub\insrsid10373936\charrsid10373936 m }{
\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid10373936 for each primer above 60}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid10373936\charrsid10373936 \'b0}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid10373936 
C and approximately match the }{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid10373936\charrsid10373936 T}{\rtlch\fcs1 \af1 \ltrch\fcs0 \sub\chshdng0\chcfpat0\chcbpat8\insrsid10373936\charrsid10373936 m}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid10373936  of paired primers}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid10373936\charrsid10373936 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af31507\afs22 \ltrch\fcs0 \f3\fs22\insrsid10373936\charrsid10373936 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls5\adjustright\rin0\lin720\itap0\pararsid10373936\contextualspace {\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid10373936\charrsid10373936 Ensure that}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid10373936 
 there is only 1 binding site on the plasmid for each primer
\par }\pard\plain \ltrpar\ql \li0\ri0\sa160\sl259\slmult1\widctlpar\wrapdefault\aspalpha\aspnum\faauto\adjustright\rin0\lin0\itap0 \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {\rtlch\fcs1 
\af31507 \ltrch\fcs0 \insrsid5784579 6/5/15
\par Continue designing primers as on 6/4 and check for c}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid10373936 orrect amplification and for suggested annealing temperatures on amplifX:
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af31507\afs22 \ltrch\fcs0 \f3\fs22\insrsid10373936 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard\plain \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls6\adjustright\rin0\lin720\itap0\pararsid10373936\contextualspace \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {\rtlch\fcs1 \af31507 
\ltrch\fcs0 \insrsid10373936 Add the primers designed in SnapGene to the primers list of AmplifX
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af31507\afs22 \ltrch\fcs0 \f3\fs22\insrsid10373936 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Add the full sequence of the plasmid to the sequence portion of AmplifX
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af31507\afs22 \ltrch\fcs0 \f3\fs22\insrsid10373936 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Select the primers in pairs as they should be used and select the full sequence; then click \'93PCR\'94 > \'93Run PCR
\'94
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af31507\afs22 \ltrch\fcs0 \f3\fs22\insrsid10373936 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Click on the amplicon in the window at the bottom to display information on it, then see the \'93Infos\'94
 tab for details
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid10373936 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls6\ilvl1\adjustright\rin0\lin1440\itap0\pararsid10373936\contextualspace {\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid10373936 
Check for expected annealing of primers, correct amplicon (length and portion of plasmid), and take note of information especially suggested annealing temperature
\par }\pard\plain \ltrpar\ql \li0\ri0\sa160\sl259\slmult1\widctlpar\wrapdefault\aspalpha\aspnum\faauto\adjustright\rin0\lin0\itap0 \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {\rtlch\fcs1 
\af31507 \ltrch\fcs0 \insrsid10373936 Order primers}{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid5784579 
\par }{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid9857289 
\par 6/8/15
\par Waiting for primers\emdash no wet lab work
\par 
\par 6/9/15
\par Group the 14 amplicons to be overexpressed by size so that similarly sized fragments can be run through PCR together }{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid2718437 
\par }{\rtlch\fcs1 \af31507 \ltrch\fcs0 \insrsid6375002 Primers arrive
\par Resuspend the dry primers in Tris-EDTA (TE) solution:
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af31507\afs22 \ltrch\fcs0 \f3\fs22\insrsid6375002 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard\plain \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls8\adjustright\rin0\lin720\itap0\pararsid6375002\contextualspace \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {\rtlch\fcs1 \af31507 
\ltrch\fcs0 \insrsid6375002 Add TE in }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid6375002\charrsid6375002 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid6375002\charrsid6375002 
L 10 times the number of nmoles of primer that there are in each vial}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid6375002 \emdash just add the solution; do not pipette in and out
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid6375002 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Place the vials in the thermomixer at 65}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid6375002\charrsid5784579 \'b0C}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid6375002  for ~10 minutes
\par }\pard\plain \ltrpar\ql \li0\ri0\sa160\sl259\slmult1\widctlpar\wrapdefault\aspalpha\aspnum\faauto\adjustright\rin0\lin0\itap0\pararsid6375002 \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid6375002 Make a 10x dilution of the concentrated primer solution:
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid6375002 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard\plain \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls9\adjustright\rin0\lin720\itap0\pararsid6375002\contextualspace \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid6375002 To labeled vials, add:
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid6375002 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls9\ilvl1\adjustright\rin0\lin1440\itap0\pararsid6375002\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid6375002 90 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid6375002\charrsid6375002 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid6375002\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid6375002  of H}{
\rtlch\fcs1 \af1 \ltrch\fcs0 \sub\chshdng0\chcfpat0\chcbpat8\insrsid6375002 2}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid6375002 0 MQft
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid6375002 \hich\af2\dbch\af31506\loch\f2 o\tab}10 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid6375002\charrsid6375002 \'ec}{\rtlch\fcs1 
\af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid6375002\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid6375002  of primer solution
\par }\pard\plain \ltrpar\ql \li0\ri0\sa160\sl259\slmult1\widctlpar\wrapdefault\aspalpha\aspnum\faauto\adjustright\rin0\lin0\itap0\pararsid15891047 \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 
{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437 Picked one of the groupings of amplicons made earlier to run PCR at the same settings: today, doing hesA, nifB, and nifV. Run 100 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437  PCR tubes

\par Calculate volumes of solutions to add for 100 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 L}{
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437  tube, then make 320 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437  of solution, without adding primers or polymerase, then complete the PCR solutions in 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2718437 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard\plain \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls9\adjustright\rin0\lin720\itap0\pararsid2718437\contextualspace \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid2718437 100 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 L}{
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437  recipe:
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2718437 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls9\ilvl1\adjustright\rin0\lin1440\itap0\pararsid2718437\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437 29.9 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437  water
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2718437 \hich\af2\dbch\af31506\loch\f2 o\tab}20 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 \'ec}{\rtlch\fcs1 
\af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437  Phusion buffer 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2718437 \hich\af2\dbch\af31506\loch\f2 o\tab}16 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 \'ec}{\rtlch\fcs1 
\af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437  dNTP solution
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \ab\af2\afs22 \ltrch\fcs0 \f2\fs22\cf1\insrsid2718437 \hich\af2\dbch\af31506\loch\f2 o\tab}}{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \cf1\chshdng0\chcfpat0\chcbpat8\insrsid2718437 2 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437  forward primer

\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2718437 \hich\af2\dbch\af31506\loch\f2 o\tab}2 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 \'ec}{\rtlch\fcs1 
\af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437  reverse primer
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2718437 \hich\af2\dbch\af31506\loch\f2 o\tab}2 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 \'ec}{\rtlch\fcs1 
\af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437  22.74 ng/}{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437 
 PSL2397 (plasmid as DNA template)
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2718437 \hich\af2\dbch\af31506\loch\f2 o\tab}7.5 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 \'ec}{
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437  DMSO 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2718437 \hich\af2\dbch\af31506\loch\f2 o\tab}20 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 \'ec}{\rtlch\fcs1 
\af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437  betaine
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2718437 \hich\af2\dbch\af31506\loch\f2 o\tab}.6 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 \'ec}{\rtlch\fcs1 
\af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437  Phusion polymerase
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2718437 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls9\adjustright\rin0\lin720\itap0\pararsid2718437\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437 Make }{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid590140 PCR master mix for }{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437 320 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid590140\charrsid6375002 \'ec}{
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid590140\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid590140  }{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437 
of solution without primers or polymerase, adding:
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2718437 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls9\ilvl1\adjustright\rin0\lin1440\itap0\pararsid2718437\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437 95.68 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437  water
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2718437 \hich\af2\dbch\af31506\loch\f2 o\tab}64 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 \'ec}{\rtlch\fcs1 
\af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437  buffer
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2718437 \hich\af2\dbch\af31506\loch\f2 o\tab}51.2 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 \'ec}{
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437  dNTP solution
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2718437 \hich\af2\dbch\af31506\loch\f2 o\tab}6.4 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 \'ec}{
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437  template DNA
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2718437 \hich\af2\dbch\af31506\loch\f2 o\tab}24 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 \'ec}{\rtlch\fcs1 
\af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437  DMSO
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2718437 \hich\af2\dbch\af31506\loch\f2 o\tab}64 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 \'ec}{\rtlch\fcs1 
\af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437  betaine
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2718437 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls9\adjustright\rin0\lin720\itap0\pararsid2718437\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437 Add 95.4 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437 
 of that solution to each }{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid10506450 of 3 labeled PCR tubes, then add the following to complete the reaction mixtures}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437 
:
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2718437 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls9\ilvl1\adjustright\rin0\lin1440\itap0\pararsid2718437\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437 2 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437  }{\rtlch\fcs1 
\af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid3040168 of 10x diluted }{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437 appropriate forward primer
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2718437 \hich\af2\dbch\af31506\loch\f2 o\tab}2 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 \'ec}{\rtlch\fcs1 
\af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid3040168  of 10x diluted}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437 
 appropriate reverse primer
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2718437 \hich\af2\dbch\af31506\loch\f2 o\tab}.6 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 \'ec}{\rtlch\fcs1 
\af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2718437  polymerase
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid10506450 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls9\adjustright\rin0\lin720\itap0\pararsid2718437\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid10506450 Run the 3 PCR tubes under the following conditions:}{\rtlch\fcs1 
\af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid6375002 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid10506450 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls9\ilvl1\adjustright\rin0\lin1440\itap0\pararsid10506450\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid10506450 98}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid10506450\charrsid5784579 \'b0C}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid10506450  for 2 minutes initially
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid10506450 \hich\af2\dbch\af31506\loch\f2 o\tab}Cycle the following 35 times:
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f10\fs22\insrsid10506450 \loch\af10\dbch\af31506\hich\f10 \'a7\tab}}\pard \ltrpar\s15\ql \fi-360\li2160\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls9\ilvl2\adjustright\rin0\lin2160\itap0\pararsid10506450\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid10506450 98}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid10506450\charrsid5784579 \'b0C}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid10506450  for 25 seconds
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f10\fs22\insrsid10506450 \loch\af10\dbch\af31506\hich\f10 \'a7\tab}56}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid10506450\charrsid5784579 \'b0C}{\rtlch\fcs1 \af1 
\ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid10506450  for 35 seconds
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f10\fs22\insrsid10506450 \loch\af10\dbch\af31506\hich\f10 \'a7\tab}72}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid10506450\charrsid5784579 \'b0C}{\rtlch\fcs1 \af1 
\ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid10506450  for 45 seconds
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid10506450 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls9\ilvl1\adjustright\rin0\lin1440\itap0\pararsid10506450\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid10506450 Hold at 10}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid10506450\charrsid5784579 \'b0C}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid10506450 
\par }\pard\plain \ltrpar\ql \li0\ri0\sa160\sl259\slmult1\widctlpar\wrapdefault\aspalpha\aspnum\faauto\adjustright\rin0\lin0\itap0\pararsid10506450 \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 
{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid10506450 Prepare a gel:
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid10506450 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard\plain \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls10\adjustright\rin0\lin720\itap0\pararsid10506450\contextualspace \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {\rtlch\fcs1 \af1 
\ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid10506450 Add ~50 mL of 1% agarose (in tris acetic acid EDTA or TAE buffer) to a flask in the ethidium bromide (EtBr) area of the lab
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid10506450 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Add 4 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid10506450\charrsid6375002 \'ec}{
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid10506450\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid10506450  of EtBr to the flask and swirl to mix
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid10506450 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Pour the mixture into the gel plate, with a plastic piece inserted to form 3 large wells and a small one on the side 

\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid10506450 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls10\ilvl1\adjustright\rin0\lin1440\itap0\pararsid10506450\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid10506450 Try to remove bubbles\emdash 
can chase them out with a pipette tip
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid10506450 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls10\adjustright\rin0\lin720\itap0\pararsid10506450\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid10506450 Allow the gel to set
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid10506450 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Pour additional TAE buffer into the plate as necessary 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid10506450 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Pipette in and out of wells to clear them of any agarose\emdash want clean wells in the gel
\par }\pard\plain \ltrpar\ql \li0\ri0\sa160\sl259\slmult1\widctlpar\wrapdefault\aspalpha\aspnum\faauto\adjustright\rin0\lin0\itap0\pararsid10506450 \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 
{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid10506450 Take samples out of PCR thermocycler and run them in the gel:
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid10506450 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard\plain \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls11\adjustright\rin0\lin720\itap0\pararsid10506450\contextualspace \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {\rtlch\fcs1 \af1 
\ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid10506450 Add 15 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid10506450\charrsid6375002 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid10506450\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid10506450  of 10x DNA loading dye to each and pipette in and out to mix to homogeneity
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid10506450 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Add each mixture to one of the wells in the gel
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid10506450 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Add 1 kb ladder to the small well
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid10506450 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Run the gel at ~82 V for ~20 minutes until 2 different shades of blue bands appear
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid10506450 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Run the gel at ~120 V for an additional ~20 minutes 
\par }\pard\plain \ltrpar\ql \li0\ri0\sa160\sl259\slmult1\widctlpar\wrapdefault\aspalpha\aspnum\faauto\adjustright\rin0\lin0\itap0\pararsid590140 \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid590140 
\par 6/10/15
\par }\pard \ltrpar\ql \li0\ri0\sa160\sl259\slmult1\widctlpar\tx4020\wrapdefault\aspalpha\aspnum\faauto\adjustright\rin0\lin0\itap0\pararsid590140 {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid590140 Run PCR for the remaining amplicons\tab }
{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid590140\charrsid590140 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid590140 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard\plain \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls9\adjustright\rin0\lin720\itap0\pararsid590140\contextualspace \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid590140 Make PCR master mix for 1200 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid590140\charrsid6375002 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid590140\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid590140  of solution without primers or polymerase, adding:
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid590140 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls9\ilvl1\adjustright\rin0\lin1440\itap0\pararsid590140\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid590140 358.8 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid590140\charrsid6375002 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid590140\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid590140  water (add 359 }{
\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid590140\charrsid6375002 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid590140\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid590140  with a P1000)
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid590140 \hich\af2\dbch\af31506\loch\f2 o\tab}240 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid590140\charrsid6375002 \'ec}{\rtlch\fcs1 
\af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid590140\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid590140  buffer
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid590140 \hich\af2\dbch\af31506\loch\f2 o\tab}192 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid590140\charrsid6375002 \'ec}{\rtlch\fcs1 
\af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid590140\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid590140  dNTP solution
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid590140 \hich\af2\dbch\af31506\loch\f2 o\tab}24 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid590140\charrsid6375002 \'ec}{\rtlch\fcs1 
\af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid590140\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid590140  22.74 ng/}{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid590140\charrsid6375002 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid590140\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid590140 
 PSL2397 (plasmid as DNA template)
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid590140 \hich\af2\dbch\af31506\loch\f2 o\tab}90 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid590140\charrsid6375002 \'ec}{\rtlch\fcs1 
\af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid590140\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid590140  DMSO
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid590140 \hich\af2\dbch\af31506\loch\f2 o\tab}240 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid590140\charrsid6375002 \'ec}{\rtlch\fcs1 
\af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid590140\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid590140  betaine
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid590140 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls9\adjustright\rin0\lin720\itap0\pararsid590140\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid590140 Add 95.4 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid590140\charrsid6375002 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid590140\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid590140 
 of master mix to each of 11 labeled PCR tubes
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid3040168 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls9\adjustright\rin0\lin720\itap0\pararsid3040168\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid3040168 Add 95.4 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid3040168\charrsid6375002 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid3040168\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid3040168 
 of that solution to each of 11 labeled PCR tubes, then add the following to complete the reaction mixtures:
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid3040168 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls9\ilvl1\adjustright\rin0\lin1440\itap0\pararsid3040168\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid3040168 2 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid3040168\charrsid6375002 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid3040168\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid3040168 
 of 10x diluted appropriate forward primer
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid3040168 \hich\af2\dbch\af31506\loch\f2 o\tab}2 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid3040168\charrsid6375002 \'ec}{\rtlch\fcs1 
\af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid3040168\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid3040168  of 10x diluted appropriate reverse primer
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid3040168 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls9\ilvl1\adjustright\rin0\lin1440\itap0\pararsid2884371\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid3040168 .6 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid3040168\charrsid6375002 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid3040168\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid3040168  polymerase}{
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2884371 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2884371 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls9\adjustright\rin0\lin720\itap0\pararsid2884371\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2884371 Thermocycler conditions for largest size group (nifHDK and nifEN):

\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2884371 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls9\ilvl1\adjustright\rin0\lin1440\itap0\pararsid2884371\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2884371 98}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid2884371\charrsid5784579 \'b0C}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2884371  for 2 minutes initially
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2884371 \hich\af2\dbch\af31506\loch\f2 o\tab}Cycle the following 35 times:
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f10\fs22\insrsid2884371 \loch\af10\dbch\af31506\hich\f10 \'a7\tab}}\pard \ltrpar\s15\ql \fi-360\li2160\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls9\ilvl2\adjustright\rin0\lin2160\itap0\pararsid2884371\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2884371 98}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid2884371\charrsid5784579 \'b0C}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2884371  for 25 seconds
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f10\fs22\insrsid2884371 \loch\af10\dbch\af31506\hich\f10 \'a7\tab}56}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2884371\charrsid5784579 \'b0C}{\rtlch\fcs1 \af1 
\ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2884371  for 35 seconds
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f10\fs22\insrsid2884371 \loch\af10\dbch\af31506\hich\f10 \'a7\tab}72}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2884371\charrsid5784579 \'b0C}{\rtlch\fcs1 \af1 
\ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2884371  for 2 minutes and 30 seconds
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2884371 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls9\ilvl1\adjustright\rin0\lin1440\itap0\pararsid2884371\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2884371 72}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid2884371\charrsid5784579 \'b0C}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2884371  for 10 minutes (final extension time)
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2884371 \hich\af2\dbch\af31506\loch\f2 o\tab}Hold at 10\'b0C
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2884371 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls9\adjustright\rin0\lin720\itap0\pararsid2884371\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2884371 Thermocycler conditions for smallest size group:
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2884371 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls9\ilvl1\adjustright\rin0\lin1440\itap0\pararsid2884371\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2884371 98}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid2884371\charrsid5784579 \'b0C}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2884371  for 2 minutes initially
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2884371 \hich\af2\dbch\af31506\loch\f2 o\tab}Cycle the following 35 times:
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f10\fs22\insrsid2884371 \loch\af10\dbch\af31506\hich\f10 \'a7\tab}}\pard \ltrpar\s15\ql \fi-360\li2160\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls9\ilvl2\adjustright\rin0\lin2160\itap0\pararsid2884371\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2884371 98}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid2884371\charrsid5784579 \'b0C}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2430266  for 20}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2884371  seconds
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f10\fs22\insrsid2430266 \loch\af10\dbch\af31506\hich\f10 \'a7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2430266 55}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid2884371\charrsid5784579 \'b0C}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2430266  for 25}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2884371  seconds
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f10\fs22\insrsid2884371 \loch\af10\dbch\af31506\hich\f10 \'a7\tab}72}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2884371\charrsid5784579 \'b0C}{\rtlch\fcs1 \af1 
\ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2884371  for }{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2430266 25 seconds}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2884371 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2884371 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls9\ilvl1\adjustright\rin0\lin1440\itap0\pararsid2884371\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2884371 72}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid2884371\charrsid5784579 \'b0C}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2884371  for 10 minutes (final extension time)
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2884371 \hich\af2\dbch\af31506\loch\f2 o\tab}Hold at 10\'b0C
\par }\pard\plain \ltrpar\ql \li0\ri0\sa160\sl259\slmult1\widctlpar\wrapdefault\aspalpha\aspnum\faauto\adjustright\rin0\lin0\itap0\pararsid2430266 \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2430266 Prepare gels for PCR products with 1% agarose
\par Dilute concentrated DNA ladder (.5 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2430266\charrsid6375002 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2430266 g/}{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2430266\charrsid6375002 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2430266\charrsid6375002 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2430266  1kb plus):

\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2430266 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard\plain \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls13\adjustright\rin0\lin720\itap0\pararsid2430266\contextualspace \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid2430266 Combine:
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2430266\charrsid2430266 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls13\ilvl1\adjustright\rin0\lin1440\itap0\pararsid2430266\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2430266\charrsid2430266 40 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2430266\charrsid2430266 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2430266\charrsid2430266 L DNA}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2430266 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2430266\charrsid2430266 \hich\af2\dbch\af31506\loch\f2 o\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2430266\charrsid2430266 40 }{\rtlch\fcs1 
\ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2430266\charrsid2430266 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2430266\charrsid2430266 L water}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid2430266 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2430266\charrsid2430266 \hich\af2\dbch\af31506\loch\f2 o\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2430266\charrsid2430266 40 }{\rtlch\fcs1 
\ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2430266\charrsid2430266 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2430266\charrsid2430266 L DNA loading dye }{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid2430266 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2430266\charrsid2430266 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls13\adjustright\rin0\lin720\itap0\pararsid2884371\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2430266\charrsid2430266 Pipette in and out to mix}{\rtlch\fcs1 \af1 
\ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2884371 
\par }\pard\plain \ltrpar\ql \li0\ri0\sa160\sl259\slmult1\widctlpar\wrapdefault\aspalpha\aspnum\faauto\adjustright\rin0\lin0\itap0\pararsid2784689 \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2784689 Add 12 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2784689\charrsid2430266 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid2784689\charrsid2430266 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2784689  of DNA loading dye to each 100 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2784689\charrsid2430266 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2784689\charrsid2430266 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2784689 
 PCR reaction tube
\par Run gels, cut out bands
\par Purify the PCR product, using a kit:
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2784689 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard\plain \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls23\adjustright\rin0\lin720\itap0\pararsid2784689\contextualspace \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid2784689 Spin down the bands in vials
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2784689 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Add BNL buffer to cover the gel
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2784689 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Place on thermomixer at 65\'b0C for about 10 minutes until the agarose is no longer solid
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2784689 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Split into 2 tubes per PCR product
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2784689 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Add 150 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2784689\charrsid2430266 \'ec}{
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2784689\charrsid2430266 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2784689  of isopropanol and invert to mix
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2784689 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Place one spin column in a collection tube for each DNA gel mixture
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2784689 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Add 750 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2784689\charrsid2430266 \'ec}{
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2784689\charrsid2430266 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2784689 
 of DNA mixture to the column and centrifuge at 13000 rpm for 1 minute; discard the flow-through and place back in same collection tube
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2784689 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Repeat the previous step with addition DNA mix until all the mix has run through the column
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2784689 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Add 700 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2784689\charrsid2430266 \'ec}{
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2784689\charrsid2430266 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2784689 
 of washing buffer to the column and spin at 13000 rpm for 1 minute; discard the flow through and replace in collection tube
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2784689 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Repeat previous step with additional 700 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2784689\charrsid2430266 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2784689\charrsid2430266 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2784689 
 of washing buffer
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2784689 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Centrifuge for 2 minutes at 13,000 rpm 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2784689 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Place columns in thermomixer for ~3 minutes
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2784689 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Place columns into a clean 1.5 mL microcentrifuge tube and apply 30 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2784689\charrsid2430266 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2784689\charrsid2430266 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2784689 
 of water to the center of the column to elute
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2784689 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Incubate at room temperature for 1 minute
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2784689 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Centrifuge for 1 minute at 13,000 rpm
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2784689 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls23\adjustright\rin0\lin720\itap0\pararsid2884371\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2784689 Measure concentrations on nanodrop with 1.5 }{\rtlch\fcs1 \ab\af1 
\ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2784689\charrsid2430266 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2784689\charrsid2430266 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2784689 
 of each sample}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2884371\charrsid2784689 
\par }\pard\plain \ltrpar\ql \li0\ri0\sa160\sl259\slmult1\widctlpar\wrapdefault\aspalpha\aspnum\faauto\adjustright\rin0\lin0\itap0\pararsid2884371 \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2884371 6/11/15
\par }{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid8921509 Perform a restriction enzyme digest of PCR products and of the plasmid backbone using 20 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid8921509\charrsid2430266 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid8921509\charrsid2430266 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid8921509  reactions:

\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid8921509 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard\plain \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls14\adjustright\rin0\lin720\itap0\pararsid8921509\contextualspace \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid8921509 Recipe specifications:
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid8921509 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls14\ilvl1\adjustright\rin0\lin1440\itap0\pararsid8921509\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid8921509 Use 2 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid8921509\charrsid2430266 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid8921509\charrsid2430266 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid8921509 
 10x buffer with dye
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid8921509 \hich\af2\dbch\af31506\loch\f2 o\tab}Enough volume of PCR product to provide 500 ng if possible (300 is enough if the concentration is too low)
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid8921509 \hich\af2\dbch\af31506\loch\f2 o\tab}1 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid8921509\charrsid2430266 \'ec}{\rtlch\fcs1 
\af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid8921509\charrsid2430266 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid8921509  of each restriction enzyme (XhoI and EcoRI as appropriate)
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid8921509 \hich\af2\dbch\af31506\loch\f2 o\tab}Water to reach 20 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid8921509\charrsid2430266 
\'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid8921509\charrsid2430266 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid8921509  total volume
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid8921509 \hich\af2\dbch\af31506\loch\f2 o\tab}Order of addition: water, buffer, DNA, enzyme
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid8921509 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls14\adjustright\rin0\lin720\itap0\pararsid8921509\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid8921509 Centrifuge for 1 minute; incubate at 37\'b0C for ~3 hours
\par }\pard\plain \ltrpar\ql \li0\ri0\sa160\sl259\slmult1\widctlpar\wrapdefault\aspalpha\aspnum\faauto\adjustright\rin0\lin0\itap0\pararsid8921509 \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid8921509 Redo PCR for the gene that did not have a band, using the same recipe and conditions as previously used
\par Prepare two gels:
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid8921509 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard\plain \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls15\adjustright\rin0\lin720\itap0\pararsid8921509\contextualspace \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid8921509 1 for restriction digest products with 1% agarose
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid13704892 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls15\ilvl1\adjustright\rin0\lin1440\itap0\pararsid13704892\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid13704892 Add ladder and plasmid digest 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f10\fs22\insrsid13704892 \loch\af10\dbch\af31506\hich\f10 \'a7\tab}}\pard \ltrpar\s15\ql \fi-360\li2160\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls15\ilvl2\adjustright\rin0\lin2160\itap0\pararsid13704892\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid13704892 Can differentiate between digested 
and undigested (or linearized) plasmid, but other products have too small of a difference between digested and undigested to tell
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid8921509 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls15\adjustright\rin0\lin720\itap0\pararsid8921509\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid8921509 1 for PCR products of the small gene for which PCR is being redone

\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid15010177 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls15\ilvl1\adjustright\rin0\lin1440\itap0\pararsid15010177\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid15010177 Make 2% agarose (helpful for smaller bands): 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f10\fs22\insrsid15010177 \loch\af10\dbch\af31506\hich\f10 \'a7\tab}}\pard \ltrpar\s15\ql \fi-360\li2160\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls15\ilvl2\adjustright\rin0\lin2160\itap0\pararsid15010177\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid15010177 Weigh 1 g agarose
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f10\fs22\insrsid15010177 \loch\af10\dbch\af31506\hich\f10 \'a7\tab}Add 50 mL TAE buffer
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f10\fs22\insrsid15010177 \loch\af10\dbch\af31506\hich\f10 \'a7\tab}Microwave until all agarose is dissolved 
\par }\pard\plain \ltrpar\ql \li0\ri0\sa160\sl259\slmult1\widctlpar\wrapdefault\aspalpha\aspnum\faauto\adjustright\rin0\lin0\itap0\pararsid8921509 \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid13704892 Purify the DNA fragments from the other digests (non-plasmid) using the small kit to elute at higher concentration:}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid8921509 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid13704892 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard\plain \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls16\adjustright\rin0\lin720\itap0\pararsid13704892\contextualspace \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {\rtlch\fcs1 \af1 
\ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid13704892 Add 100 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid13704892\charrsid2430266 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid13704892\charrsid2430266 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid13704892  of DNA binding buffer to each sample
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid13704892 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls16\ilvl1\adjustright\rin0\lin1440\itap0\pararsid13704892\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid13704892 20 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid13704892\charrsid2430266 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid13704892\charrsid2430266 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid13704892  }{
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid14615753 digest reaction x 5 (multiplier for DNA fragments, according to kit) = 10 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid14615753\charrsid2430266 
\'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid14615753\charrsid2430266 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid14615753  to add}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid13704892 

\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid14615753 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls16\adjustright\rin0\lin720\itap0\pararsid14615753\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid14615753 Pipette in and out to mix
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid14615753 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Transfer mixture to a spin column in a labeled collection tube
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid14615753 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Centrifuge for 30 seconds; discard the flow-through
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid14615753 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Add 200 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid14615753\charrsid2430266 \'ec}{
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid14615753\charrsid2430266 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid14615753  of DNA wash buffer to the column. Centrifuge for 30 seconds. Repeat the wash step
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid14615753 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Add 10 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid14615753\charrsid2430266 \'ec}{
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid14615753\charrsid2430266 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid14615753  water to the column matrix and incubate at room temperature for ~2 minutes
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid14615753 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Transfer the column to a 1.5 mL microcentrifuge tube and centrifuge for 30 seconds to elute the DNA
\par }\pard\plain \ltrpar\ql \li0\ri0\sa160\sl259\slmult1\widctlpar\wrapdefault\aspalpha\aspnum\faauto\adjustright\rin0\lin0\itap0\pararsid14615753 \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 
{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid14615753 Quantify the eluate with the nanodrop using 1 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid14615753\charrsid2430266 \'ec}{\rtlch\fcs1 \af1 
\ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid14615753\charrsid2430266 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid14615753  of each sample 
\par }{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid5314342 Run gels for plasmid}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281  digest}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid5314342 
 and PCR product 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid5314342 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard\plain \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls17\adjustright\rin0\lin720\itap0\pararsid5314342\contextualspace \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid5314342 Plasmid:
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid5314342 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls17\ilvl1\adjustright\rin0\lin1440\itap0\pararsid5314342\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid5314342 
saw bands for fragment, backbone with fragment cut out, and linearized fragment (hoped to see only the first two)
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid5314342 \hich\af2\dbch\af31506\loch\f2 o\tab}cut out only the band of the backbone with fragment cut out and purified
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid5314342 \hich\af2\dbch\af31506\loch\f2 o\tab}took concentration on nanodrop
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid5314342 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls17\adjustright\rin0\lin720\itap0\pararsid5314342\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid5314342 PCR product:
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid5314342 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls17\ilvl1\adjustright\rin0\lin1440\itap0\pararsid5314342\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid5314342 Cut and purified band
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid5314342 \hich\af2\dbch\af31506\loch\f2 o\tab}Took concentration on nanodrop
\par }\pard\plain \ltrpar\ql \li0\ri0\sa160\sl259\slmult1\widctlpar\wrapdefault\aspalpha\aspnum\faauto\adjustright\rin0\lin0\itap0\pararsid5314342 \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281 Purify the DNA fragments from non-plasmid digests using a kid for high concentration:
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2190281 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard\plain \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls19\adjustright\rin0\lin720\itap0\pararsid2190281\contextualspace \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid2190281 Add 100 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2190281\charrsid2430266 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281\charrsid2430266 L}{
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281  of DNA binding buffer to each sample
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2190281 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls19\ilvl1\adjustright\rin0\lin1440\itap0\pararsid2190281\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281 20 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2190281\charrsid2430266 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281\charrsid2430266 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281 
 sample x 5 (multiplier for DNA fragments, according to kit) = 100 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2190281\charrsid2430266 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid2190281\charrsid2430266 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2190281 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls19\adjustright\rin0\lin720\itap0\pararsid2190281\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281 Pipette }{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid2190281\charrsid2190281 in and out to mix}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2190281\charrsid2190281 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281\charrsid2190281 
Transfer the mixture to a spin column in a labeled collection tube}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2190281\charrsid2190281 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281\charrsid2190281 
Centrifuge for 30 seconds; discard the flow-through}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2190281\charrsid2190281 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281\charrsid2190281 Add 200 }{
\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2190281\charrsid2190281 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281\charrsid2190281 
L of DNA wash buffer to the column. Centrifuge for 30 seconds}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2190281 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Repeat }{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281\charrsid2190281 the wash step}{
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2190281\charrsid2190281 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281\charrsid2190281 Ad}{\rtlch\fcs1 
\af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281 d}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281\charrsid2190281  10 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2190281\charrsid2190281 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281\charrsid2190281 L water to the column matrix and incubate at room temperature for ~2 minutes}{\rtlch\fcs1 
\af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2190281\charrsid2190281 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281\charrsid2190281 
Transfer the column to a 1.5 mL microcentrifuge tube and centrifuge for 30 seconds to elute the DNA}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2190281\charrsid2190281 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281\charrsid2190281 
Quantify concentrations with the nanodrop\emdash 1 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2190281\charrsid2190281 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281\charrsid2190281 
L of each sample}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2190281\charrsid2190281 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281\charrsid2190281 Store at }{
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281 -20\'b0C}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281\charrsid2190281 
\par }\pard\plain \ltrpar\ql \li0\ri0\sa160\sl259\slmult1\widctlpar\wrapdefault\aspalpha\aspnum\faauto\adjustright\rin0\lin0\itap0\pararsid5314342 \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid5314342 Set up 7 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid5314342\charrsid2430266 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid5314342\charrsid2430266 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281  ligation reactions for the}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid6761215 
 digestion products that were digested by both enzymes:
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2190281 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard\plain \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls18\adjustright\rin0\lin720\itap0\pararsid6761215\contextualspace \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid2190281 Recipe conditions:
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2190281 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls18\ilvl1\adjustright\rin0\lin1440\itap0\pararsid2190281\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281 .7 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2190281\charrsid2190281 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281\charrsid2190281 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281  buffer}{
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid6761215 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2190281 \hich\af2\dbch\af31506\loch\f2 o\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281 1 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2190281\charrsid2190281 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281\charrsid2190281 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281  enzyme
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2190281 \hich\af2\dbch\af31506\loch\f2 o\tab}.7 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2190281\charrsid2190281 \'ec}{\rtlch\fcs1 
\af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281\charrsid2190281 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281  digested plasmid
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2190281 \hich\af2\dbch\af31506\loch\f2 o\tab}5x more insert DNA than plasmid DNA
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2190281 \hich\af2\dbch\af31506\loch\f2 o\tab}Water to final volume
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2190281 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls18\adjustright\rin0\lin720\itap0\pararsid6761215\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281 Order of addition: water, DNA, buffer, enzyme
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2190281 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Set up an additional ligation reaction for negative control: see if the plasmid will ligate to itself with no enzyme

\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2190281 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Cyclic ligation conditions:
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2190281 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls18\ilvl1\adjustright\rin0\lin1440\itap0\pararsid2190281\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281 37\'b0C for 3 minutes
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2190281 \hich\af2\dbch\af31506\loch\f2 o\tab}22\'b0C for 3 minutes
\par }\pard\plain \ltrpar\ql \li0\ri0\sa160\sl259\slmult1\widctlpar\wrapdefault\aspalpha\aspnum\faauto\adjustright\rin0\lin0\itap0\pararsid2190281 \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281 Start an overnight culture}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid884068  in 5 mL of LB at 37\'b0C}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid2190281  for transformation of ligated plasmids
\par }{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid884068 6/12/15
\par Prepare competent cells using the RbCl method:
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid884068 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard\plain \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls20\adjustright\rin0\lin720\itap0\pararsid884068\contextualspace \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid884068 Dilute 1 mL of culture into 50 mL of LBMg medium pre-warmed to 37\'b0C
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid884068 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Grow at 37\'b0C for approximately 2 hours on shaker to OD}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\sub\chshdng0\chcfpat0\chcbpat8\insrsid884068 600}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid884068  of about 0.6
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid884068 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls20\ilvl1\adjustright\rin0\lin1440\itap0\pararsid884068\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid884068 Final OD}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\sub\chshdng0\chcfpat0\chcbpat8\insrsid884068 600 }{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid884068\charrsid884068 =}{\rtlch\fcs1 \af1 \ltrch\fcs0 \sub\chshdng0\chcfpat0\chcbpat8\insrsid884068  }{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid884068 0.598
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid884068 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls20\adjustright\rin0\lin720\itap0\pararsid884068\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid884068 Do not vortex cells after this point or allow them to warm above 4\'b0C

\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid884068 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Incubate for 20 minutes on ice
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid884068 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Transfer culture to an ice-cold 50 mL conical tube
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid884068 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Centrifuge for 15 minutes at 3000 rpm and 4\'b0C; pour of the supernatant
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid884068 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Resuspend in 20 mL of Tbf1 from 4\'b0C fridge
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid884068 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls20\ilvl1\adjustright\rin0\lin1440\itap0\pararsid884068\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid884068 
Using pipette tips from the freezer, start with less than 20 mL and pipette in and out to resuspend, then add up to 20 mL volume
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid884068 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls20\adjustright\rin0\lin720\itap0\pararsid884068\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid884068 Incubate on ice for 25 minutes 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid884068 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Centrifuge for 10 minutes at 3000 rpm and 4\'b0C; remove the supernatant
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid884068 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Resuspend in 4 mL of Tbf2 from the fridge
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid884068 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Aliquot 100 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid884068\charrsid2190281 \'ec}{
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid884068\charrsid2190281 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid884068  into microcentrifuge tubes
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid884068 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls20\ilvl1\adjustright\rin0\lin1440\itap0\pararsid884068\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid884068 
Work in the hood with cells on ice and place filled tubes into liquid nitrogen
\par }\pard\plain \ltrpar\ql \li0\ri0\sa160\sl259\slmult1\widctlpar\wrapdefault\aspalpha\aspnum\faauto\adjustright\rin0\lin0\itap0\pararsid884068 \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid884068 Transform products from yesterday\rquote s ligation reactions into the cells:
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid884068 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard\plain \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls21\adjustright\rin0\lin720\itap0\pararsid884068\contextualspace \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid884068 Use PE5A plasmid (with ampicillin resistance) as an additional control
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid884068 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls21\ilvl1\adjustright\rin0\lin1440\itap0\pararsid884068\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid884068 Original concentration of plasmid: 375 ng/}{\rtlch\fcs1 \ab\af1 
\ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid884068\charrsid2190281 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid884068\charrsid2190281 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid884068 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid884068 \hich\af2\dbch\af31506\loch\f2 o\tab}Take 2 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid884068\charrsid2190281 \'ec}{
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid884068\charrsid2190281 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid884068  of PE5A and 98 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid884068\charrsid2190281 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid884068\charrsid2190281 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid884068  water
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid13771675 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls21\ilvl1\adjustright\rin0\lin1440\itap0\pararsid13771675\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid13771675 Take 1 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid13771675\charrsid2190281 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid13771675\charrsid2190281 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid13771675 
 of that mixture and add 99 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid13771675\charrsid2190281 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid13771675\charrsid2190281 L}{\rtlch\fcs1 \af1 
\ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid13771675  water
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid13771675 \hich\af2\dbch\af31506\loch\f2 o\tab}Add 1 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid13771675\charrsid2190281 \'ec}{
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid13771675\charrsid2190281 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid13771675  of that to a vial of competent cells
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid13771675 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls21\adjustright\rin0\lin720\itap0\pararsid13771675\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid13771675 
Add the ligation reaction products to other labeled vials of competent cells 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid13771675 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Heat shock at 42\'b0C for 1 minute; replace on ice for ~2 minutes
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid13771675 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Add 900 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid13771675\charrsid2190281 \'ec}{
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid13771675\charrsid2190281 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid14826441  }{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid13771675 
of LB to each tube (working within the radius of flame)
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid13771675 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Place on shaker at 37\'b0C for 1 hour
\par }\pard\plain \ltrpar\ql \li0\ri0\sa160\sl259\slmult1\widctlpar\wrapdefault\aspalpha\aspnum\faauto\adjustright\rin0\lin0\itap0\pararsid13771675 \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 
{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid13771675 Make plates:
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid13771675 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard\plain \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls22\adjustright\rin0\lin720\itap0\pararsid13771675\contextualspace \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {\rtlch\fcs1 \af1 
\ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid13771675 Melt LB agar in the microwave; incubate on ice for 25 minutes
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid13771675 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Add appropriate antibiotic to LB agar
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid13771675 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls22\ilvl1\adjustright\rin0\lin1440\itap0\pararsid13771675\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid13771675 Antibiotic is heat sensitive\emdash wait until cooled on ice

\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid13771675 \hich\af2\dbch\af31506\loch\f2 o\tab}Antibiotic stocks in the lab are prepared such that 1 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid13771675\charrsid2190281 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid13771675\charrsid2190281 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid13771675 
 is needed per mL of agar
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid13771675 \hich\af2\dbch\af31506\loch\f2 o\tab}Use chloramphenicol for the digests and negative control
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid13771675 \hich\af2\dbch\af31506\loch\f2 o\tab}Use ampicillin for the PE5A plasmid
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid13771675 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls22\adjustright\rin0\lin720\itap0\pararsid13771675\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid13771675 
Pour ~20 mL of LB agar with antibiotic per plate; pour slowly to avoid bubbles
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid13771675 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Place in hood with lids slightly off to avoid condensation
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2784689 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2784689 
Label bottom of plates with antibiotic, gene, and date}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid13771675 
\par }\pard\plain \ltrpar\ql \li0\ri0\sa160\sl259\slmult1\widctlpar\wrapdefault\aspalpha\aspnum\faauto\adjustright\rin0\lin0\itap0\pararsid2784689 \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2784689 Plate cells:
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2784689 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard\plain \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls24\adjustright\rin0\lin720\itap0\pararsid2784689\contextualspace \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid2784689 Take cells out of 37\'b0C room}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid6060177  }{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2784689 
and spin down all but the control for efficiency of transformation (PE5A) for 4 minutes
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid2784689 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}Take 100 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2784689\charrsid2190281 \'ec}{
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2784689\charrsid2190281 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2784689  from PE5A control and add to 900 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2784689\charrsid2190281 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2784689\charrsid2190281 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2784689  of LB
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2784689 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls24\ilvl1\adjustright\rin0\lin1440\itap0\pararsid2784689\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2784689 Plate 100 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid2784689\charrsid2190281 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2784689\charrsid2190281 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2784689 
 of that dilution on the amp plate
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2784689 \hich\af2\dbch\af31506\loch\f2 o\tab}Add sterile glass beads and shake laterally to spread around the culture
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid2784689 \hich\af2\dbch\af31506\loch\f2 o\tab}Dump beads into nonsterile glass beads container
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid6060177 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls24\adjustright\rin0\lin720\itap0\pararsid2784689\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid6060177 From centrifuged cultures, pipette off the media to the 100 }{
\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid6060177\charrsid2190281 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid6060177\charrsid2190281 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid6060177  mark and resuspend the pellet in that amount of media}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2784689 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid6060177 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls24\ilvl1\adjustright\rin0\lin1440\itap0\pararsid6060177\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid6060177 Add full resuspended quantity to the correct labeled plate

\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid6060177 \hich\af2\dbch\af31506\loch\f2 o\tab}Add beads and shake
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid6060177 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls24\adjustright\rin0\lin720\itap0\pararsid6060177\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid6060177 Place all plates in 37\'b0C room
\par }\pard\plain \ltrpar\ql \li0\ri0\sa160\sl259\slmult1\widctlpar\wrapdefault\aspalpha\aspnum\faauto\adjustright\rin0\lin0\itap0\pararsid6060177 \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {
\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid6060177 6/13/15
\par Take plates out of 37\'b0C room, check for colonies, and place those that grew into the 4\'b0C room for the remainder of the weekend
\par 6/15/15
\par Start a culture of PA2C-TesA in LB for ~4 hours
\par Redo PCR using same conditions as previously for the genes that were not successfully transformed
\par Prepare a gel
\par Run the PCR products on the gel and cut appropriate bands
\par Purify the PCR product with a kit, as previously and take concentrations on nanodrop
\par Purify the PA2C-TesA plasmid as previously and take concentration on nanodrop
\par Set up 20 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid6060177\charrsid2190281 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid6060177\charrsid2190281 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid6060177  digest re}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid14826441 actions for the newly purified genes using the same specifications as previously 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid14826441 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard\plain \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls25\adjustright\rin0\lin720\itap0\pararsid14826441\contextualspace \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {\rtlch\fcs1 \af1 
\ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid14826441 Place at 37\'b0C for ~2 hours
\par }\pard\plain \ltrpar\ql \li0\ri0\sa160\sl259\slmult1\widctlpar\wrapdefault\aspalpha\aspnum\faauto\adjustright\rin0\lin0\itap0\pararsid14826441 \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 
{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid14826441 Prepare a gel
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid14826441\charrsid14826441 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard\plain \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls25\adjustright\rin0\lin720\itap0\pararsid14826441\contextualspace \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 {\rtlch\fcs1 \af1 
\ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid14826441\charrsid14826441 ~50 mL 1% agarose and 4}{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid14826441\charrsid14826441  \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid14826441\charrsid14826441 L of EtBr
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid14826441 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid14826441 Run digested plasmid through the gel 
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid14826441 \hich\af2\dbch\af31506\loch\f2 o\tab}}\pard \ltrpar\s15\ql \fi-360\li1440\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls25\ilvl1\adjustright\rin0\lin1440\itap0\pararsid14826441\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid14826441 First well: undigested plasmid (2 }{\rtlch\fcs1 \ab\af1 
\ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid14826441\charrsid2190281 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid14826441\charrsid2190281 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid14826441 
 1:10 PA2C-TesA, 2 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid14826441\charrsid2190281 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid14826441\charrsid2190281 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid14826441  DNA loading dye 10x, and 16 }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid14826441\charrsid2190281 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid14826441\charrsid2190281 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid14826441  water)
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid14826441 \hich\af2\dbch\af31506\loch\f2 o\tab}Second well: digested plasmid (3}{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 
\cf1\chshdng0\chcfpat0\chcbpat8\insrsid14826441\charrsid14826441  }{\rtlch\fcs1 \ab\af1 \ltrch\fcs0 \f31571\cf1\chshdng0\chcfpat0\chcbpat8\insrsid14826441\charrsid2190281 \'ec}{\rtlch\fcs1 \af1 \ltrch\fcs0 
\chshdng0\chcfpat0\chcbpat8\insrsid14826441\charrsid2190281 L}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid14826441  10x loading dye and digest product)
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af2\afs22 \ltrch\fcs0 \f2\fs22\insrsid14826441 \hich\af2\dbch\af31506\loch\f2 o\tab}Last well: 1 kb plus ladder
\par {\listtext\pard\plain\ltrpar \s15 \rtlch\fcs1 \af1\afs22 \ltrch\fcs0 \f3\fs22\insrsid14826441 \loch\af3\dbch\af31506\hich\f3 \'b7\tab}}\pard \ltrpar\s15\ql \fi-360\li720\ri0\sa160\sl259\slmult1
\widctlpar\wrapdefault\aspalpha\aspnum\faauto\ls25\adjustright\rin0\lin720\itap0\pararsid14826441\contextualspace {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid14826441 
Saw no band for digested plasmid and a band in unexpected location for the undigested plasmid}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid14826441\charrsid14826441 {\field{\*\fldinst SYMBOL 224 \\f "Wingdings" \\s 11}{\fldrslt
\f10\fs22}}}{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid14826441  need to redo the digestion of the plasmid
\par }\pard\plain \ltrpar\ql \li0\ri0\sa160\sl259\slmult1\widctlpar\wrapdefault\aspalpha\aspnum\faauto\adjustright\rin0\lin0\itap0\pararsid14826441 \rtlch\fcs1 \af31507\afs22\alang1025 \ltrch\fcs0 \f31506\fs22\lang1033\langfe1033\cgrid\langnp1033\langfenp1033 
{\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid14826441\charrsid14826441 
\par }\pard \ltrpar\ql \li0\ri0\sa160\sl259\slmult1\widctlpar\wrapdefault\aspalpha\aspnum\faauto\adjustright\rin0\lin0\itap0\pararsid2190281 {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2190281\charrsid2190281 
\par }\pard \ltrpar\ql \li0\ri0\sa160\sl259\slmult1\widctlpar\wrapdefault\aspalpha\aspnum\faauto\adjustright\rin0\lin0\itap0\pararsid2884371 {\rtlch\fcs1 \af1 \ltrch\fcs0 \chshdng0\chcfpat0\chcbpat8\insrsid2884371\charrsid2884371 
\par }{\*\themedata 504b030414000600080000002100e9de0fbfff0000001c020000130000005b436f6e74656e745f54797065735d2e786d6cac91cb4ec3301045f748fc83e52d4a
9cb2400825e982c78ec7a27cc0c8992416c9d8b2a755fbf74cd25442a820166c2cd933f79e3be372bd1f07b5c3989ca74aaff2422b24eb1b475da5df374fd9ad
5689811a183c61a50f98f4babebc2837878049899a52a57be670674cb23d8e90721f90a4d2fa3802cb35762680fd800ecd7551dc18eb899138e3c943d7e503b6
b01d583deee5f99824e290b4ba3f364eac4a430883b3c092d4eca8f946c916422ecab927f52ea42b89a1cd59c254f919b0e85e6535d135a8de20f20b8c12c3b0
0c895fcf6720192de6bf3b9e89ecdbd6596cbcdd8eb28e7c365ecc4ec1ff1460f53fe813d3cc7f5b7f020000ffff0300504b030414000600080000002100a5d6
a7e7c0000000360100000b0000005f72656c732f2e72656c73848fcf6ac3300c87ef85bd83d17d51d2c31825762fa590432fa37d00e1287f68221bdb1bebdb4f
c7060abb0884a4eff7a93dfeae8bf9e194e720169aaa06c3e2433fcb68e1763dbf7f82c985a4a725085b787086a37bdbb55fbc50d1a33ccd311ba548b6309512
0f88d94fbc52ae4264d1c910d24a45db3462247fa791715fd71f989e19e0364cd3f51652d73760ae8fa8c9ffb3c330cc9e4fc17faf2ce545046e37944c69e462
a1a82fe353bd90a865aad41ed0b5b8f9d6fd010000ffff0300504b0304140006000800000021006b799616830000008a0000001c0000007468656d652f746865
6d652f7468656d654d616e616765722e786d6c0ccc4d0ac3201040e17da17790d93763bb284562b2cbaebbf600439c1a41c7a0d29fdbd7e5e38337cedf14d59b
4b0d592c9c070d8a65cd2e88b7f07c2ca71ba8da481cc52c6ce1c715e6e97818c9b48d13df49c873517d23d59085adb5dd20d6b52bd521ef2cdd5eb9246a3d8b
4757e8d3f729e245eb2b260a0238fd010000ffff0300504b030414000600080000002100aa5225dfc60600008b1a0000160000007468656d652f7468656d652f
7468656d65312e786d6cec595d8bdb46147d2ff43f08bd3bfe92fcb1c41b6cd9ceb6d94d42eca4e4716c8fadc98e344633de8d0981923c160aa569e943037deb
43691b48a02fe9afd936a54d217fa17746b63c638fbb9b2585a5640d8b343af7ce997bafce1d4997afdc8fa87384134e58dc708b970aae83e3211b9178d2706f
f7bbb99aeb7081e211a22cc60d778eb97b65f7c30f2ea31d11e2083b601ff31dd4704321a63bf93c1fc230e297d814c7706dcc920809384d26f951828ec16f44
f3a542a1928f10895d274611b8bd311e932176fad2a5bbbb74dea1701a0b2e078634e949d7d8b050d8d1615122f89c0734718e106db830cf881df7f17de13a14
7101171a6e41fdb9f9ddcb79b4b330a2628bad66d7557f0bbb85c1e8b0a4e64c26836c52cff3bd4a33f3af00546ce23ad54ea553c9fc29001a0e61a52917dda7
dfaab7dafe02ab81d2438bef76b55d2e1a78cd7f798373d3973f03af40a97f6f03dfed06104503af4029dedfc07b5eb51478065e81527c65035f2d34db5ed5c0
2b5048497cb8812ef89572b05c6d061933ba6785d77daf5b2d2d9caf50500d5975c929c62c16db6a2d42f758d2058004522448ec88f9148fd110aa3840940c12
e2ec93490885374531e3305c2815ba8532fc973f4f1da988a01d8c346bc90b98f08d21c9c7e1c3844c45c3fd18bcba1ae4cdcb1fdfbc7cee9c3c7a71f2e89793
c78f4f1efd9c3a32acf6503cd1ad5e7fffc5df4f3f75fe7afeddeb275fd9f15cc7fffed367bffdfaa51d082b5d85e0d5d7cffe78f1ecd5379ffff9c3130bbc99
a0810eef930873e73a3e766eb10816a6426032c783e4ed2cfa2122ba45339e701423398bc57f478406fafa1c5164c1b5b019c13b09488c0d787576cf20dc0b93
9920168fd7c2c8001e30465b2cb146e19a9c4b0b737f164fec9327331d770ba123dbdc018a8dfc766653d05662731984d8a07993a258a0098eb170e4357688b1
6575770931e27a408609e36c2c9cbbc46921620d499f0c8c6a5a19ed9108f232b711847c1bb139b8e3b418b5adba8d8f4c24dc15885ac8f73135c27815cd048a
6c2efb28a27ac0f791086d247bf364a8e33a5c40a6279832a733c29cdb6c6e24b05e2de9d7405eec693fa0f3c84426821cda7cee23c674649b1d06218aa6366c
8fc4a18efd881f428922e7261336f80133ef10790e7940f1d674df21d848f7e96a701b9455a7b42a107965965872791533a37e7b733a4658490d08bfa1e71189
4f15f73559f7ff5b5907217df5ed53cbaa2eaaa0371362bda3f6d6647c1b6e5dbc03968cc8c5d7ee369ac53731dc2e9b0decbd74bf976ef77f2fdddbeee7772f
d82b8d06f9965bc574abae36eed1d67dfb9850da13738af7b9daba73e84ca32e0c4a3bf5cc8ab3e7b8690887f24e86090cdc2441cac64998f88488b017a229ec
ef8bae7432e10bd713ee4c19876dbf1ab6fa96783a8b0ed8287d5c2d16e5a3692a1e1c89d578c1cfc6e15143a4e84a75f50896b9576c27ea51794940dabe0d09
6d329344d942a2ba1c9441520fe610340b09b5b277c2a26e615193ee97a9da6001d4b2acc0d6c9810d57c3f53d30012378a242148f649ed2542fb3ab92f92e33
bd2d984605c03e625901ab4cd725d7adcb93ab4b4bed0c99364868e566925091513d8c87688417d52947cf42e36d735d5fa5d4a02743a1e683d25ad1a8d6fe8d
c579730d76ebda40635d2968ec1c37dc4ad9879219a269c31dc3633f1c4653a81d2eb7bc884ee0ddd95024e90d7f1e6599265cb4110fd3802bd149d520220227
0e2551c395cbcfd24063a5218a5bb104827061c9d541562e1a3948ba99643c1ee3a1d0d3ae8dc848a7a7a0f0a95658af2af3f383a5259b41ba7be1e8d819d059
720b4189f9d5a20ce0887078fb534ca33922f03a3313b255fdad35a685eceaef13550da5e3884e43b4e828ba98a77025e5191d7596c5403b5bac1902aa8564d1
080713d960f5a01add34eb1a2987ad5df7742319394d34573dd35015d935ed2a66ccb06c036bb13c5f93d7582d430c9aa677f854bad725b7bed4bab57d42d625
20e059fc2c5df70c0d41a3b69acca026196fcab0d4ecc5a8d93b960b3c85da599a84a6fa95a5dbb5b8653dc23a1d0c9eabf383dd7ad5c2d078b9af549156df3d
f44f136c700fc4a30d2f81675470954af8f09020d810f5d49e24950db845ee8bc5ad0147ce2c210df741c16f7a41c90f72859adfc97965af90abf9cd72aee9fb
e562c72f16daadd243682c228c8a7efacda50bafa2e87cf1e5458d6f7c7d89966fdb2e0d599467eaeb4a5e11575f5f8aa5ed5f5f1c02a2f3a052ead6cbf55625
572f37bb39afddaae5ea41a5956b57826abbdb0efc5abdfbd0758e14d86b9603afd2a9e52ac520c8799582a45fabe7aa5ea9d4f4aacd5ac76b3e5c6c6360e5a9
7c2c6201e155bc76ff010000ffff0300504b0304140006000800000021000dd1909fb60000001b010000270000007468656d652f7468656d652f5f72656c732f
7468656d654d616e616765722e786d6c2e72656c73848f4d0ac2301484f78277086f6fd3ba109126dd88d0add40384e4350d363f2451eced0dae2c082e8761be
9969bb979dc9136332de3168aa1a083ae995719ac16db8ec8e4052164e89d93b64b060828e6f37ed1567914b284d262452282e3198720e274a939cd08a54f980
ae38a38f56e422a3a641c8bbd048f7757da0f19b017cc524bd62107bd5001996509affb3fd381a89672f1f165dfe514173d9850528a2c6cce0239baa4c04ca5b
babac4df000000ffff0300504b01022d0014000600080000002100e9de0fbfff0000001c0200001300000000000000000000000000000000005b436f6e74656e
745f54797065735d2e786d6c504b01022d0014000600080000002100a5d6a7e7c0000000360100000b00000000000000000000000000300100005f72656c732f
2e72656c73504b01022d00140006000800000021006b799616830000008a0000001c00000000000000000000000000190200007468656d652f7468656d652f74
68656d654d616e616765722e786d6c504b01022d0014000600080000002100aa5225dfc60600008b1a00001600000000000000000000000000d6020000746865
6d652f7468656d652f7468656d65312e786d6c504b01022d00140006000800000021000dd1909fb60000001b0100002700000000000000000000000000d00900007468656d652f7468656d652f5f72656c732f7468656d654d616e616765722e786d6c2e72656c73504b050600000000050005005d010000cb0a00000000}
{\*\colorschememapping 3c3f786d6c2076657273696f6e3d22312e302220656e636f64696e673d225554462d3822207374616e64616c6f6e653d22796573223f3e0d0a3c613a636c724d
617020786d6c6e733a613d22687474703a2f2f736368656d61732e6f70656e786d6c666f726d6174732e6f72672f64726177696e676d6c2f323030362f6d6169
6e22206267313d226c743122207478313d22646b3122206267323d226c743222207478323d22646b322220616363656e74313d22616363656e74312220616363
656e74323d22616363656e74322220616363656e74333d22616363656e74332220616363656e74343d22616363656e74342220616363656e74353d22616363656e74352220616363656e74363d22616363656e74362220686c696e6b3d22686c696e6b2220666f6c486c696e6b3d22666f6c486c696e6b222f3e}
{\*\latentstyles\lsdstimax371\lsdlockeddef0\lsdsemihiddendef0\lsdunhideuseddef0\lsdqformatdef0\lsdprioritydef99{\lsdlockedexcept \lsdqformat1 \lsdpriority0 \lsdlocked0 Normal;\lsdqformat1 \lsdpriority9 \lsdlocked0 heading 1;
\lsdsemihidden1 \lsdunhideused1 \lsdqformat1 \lsdpriority9 \lsdlocked0 heading 2;\lsdsemihidden1 \lsdunhideused1 \lsdqformat1 \lsdpriority9 \lsdlocked0 heading 3;\lsdsemihidden1 \lsdunhideused1 \lsdqformat1 \lsdpriority9 \lsdlocked0 heading 4;
\lsdsemihidden1 \lsdunhideused1 \lsdqformat1 \lsdpriority9 \lsdlocked0 heading 5;\lsdsemihidden1 \lsdunhideused1 \lsdqformat1 \lsdpriority9 \lsdlocked0 heading 6;\lsdsemihidden1 \lsdunhideused1 \lsdqformat1 \lsdpriority9 \lsdlocked0 heading 7;
\lsdsemihidden1 \lsdunhideused1 \lsdqformat1 \lsdpriority9 \lsdlocked0 heading 8;\lsdsemihidden1 \lsdunhideused1 \lsdqformat1 \lsdpriority9 \lsdlocked0 heading 9;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 index 1;
\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 index 2;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 index 3;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 index 4;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 index 5;
\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 index 6;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 index 7;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 index 8;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 index 9;
\lsdsemihidden1 \lsdunhideused1 \lsdpriority39 \lsdlocked0 toc 1;\lsdsemihidden1 \lsdunhideused1 \lsdpriority39 \lsdlocked0 toc 2;\lsdsemihidden1 \lsdunhideused1 \lsdpriority39 \lsdlocked0 toc 3;
\lsdsemihidden1 \lsdunhideused1 \lsdpriority39 \lsdlocked0 toc 4;\lsdsemihidden1 \lsdunhideused1 \lsdpriority39 \lsdlocked0 toc 5;\lsdsemihidden1 \lsdunhideused1 \lsdpriority39 \lsdlocked0 toc 6;
\lsdsemihidden1 \lsdunhideused1 \lsdpriority39 \lsdlocked0 toc 7;\lsdsemihidden1 \lsdunhideused1 \lsdpriority39 \lsdlocked0 toc 8;\lsdsemihidden1 \lsdunhideused1 \lsdpriority39 \lsdlocked0 toc 9;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Normal Indent;
\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 footnote text;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 annotation text;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 header;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 footer;
\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 index heading;\lsdsemihidden1 \lsdunhideused1 \lsdqformat1 \lsdpriority35 \lsdlocked0 caption;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 table of figures;
\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 envelope address;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 envelope return;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 footnote reference;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 annotation reference;
\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 line number;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 page number;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 endnote reference;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 endnote text;
\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 table of authorities;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 macro;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 toa heading;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 List;
\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 List Bullet;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 List Number;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 List 2;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 List 3;
\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 List 4;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 List 5;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 List Bullet 2;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 List Bullet 3;
\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 List Bullet 4;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 List Bullet 5;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 List Number 2;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 List Number 3;
\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 List Number 4;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 List Number 5;\lsdqformat1 \lsdpriority10 \lsdlocked0 Title;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Closing;
\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Signature;\lsdsemihidden1 \lsdunhideused1 \lsdpriority1 \lsdlocked0 Default Paragraph Font;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Body Text;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Body Text Indent;
\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 List Continue;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 List Continue 2;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 List Continue 3;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 List Continue 4;
\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 List Continue 5;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Message Header;\lsdqformat1 \lsdpriority11 \lsdlocked0 Subtitle;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Salutation;
\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Date;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Body Text First Indent;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Body Text First Indent 2;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Note Heading;
\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Body Text 2;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Body Text 3;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Body Text Indent 2;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Body Text Indent 3;
\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Block Text;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Hyperlink;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 FollowedHyperlink;\lsdqformat1 \lsdpriority22 \lsdlocked0 Strong;
\lsdqformat1 \lsdpriority20 \lsdlocked0 Emphasis;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Document Map;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Plain Text;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 E-mail Signature;
\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 HTML Top of Form;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 HTML Bottom of Form;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Normal (Web);\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 HTML Acronym;
\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 HTML Address;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 HTML Cite;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 HTML Code;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 HTML Definition;
\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 HTML Keyboard;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 HTML Preformatted;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 HTML Sample;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 HTML Typewriter;
\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 HTML Variable;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Normal Table;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 annotation subject;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 No List;
\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Outline List 1;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Outline List 2;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Outline List 3;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table Simple 1;
\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table Simple 2;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table Simple 3;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table Classic 1;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table Classic 2;
\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table Classic 3;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table Classic 4;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table Colorful 1;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table Colorful 2;
\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table Colorful 3;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table Columns 1;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table Columns 2;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table Columns 3;
\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table Columns 4;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table Columns 5;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table Grid 1;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table Grid 2;
\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table Grid 3;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table Grid 4;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table Grid 5;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table Grid 6;
\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table Grid 7;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table Grid 8;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table List 1;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table List 2;
\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table List 3;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table List 4;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table List 5;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table List 6;
\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table List 7;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table List 8;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table 3D effects 1;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table 3D effects 2;
\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table 3D effects 3;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table Contemporary;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table Elegant;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table Professional;
\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table Subtle 1;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table Subtle 2;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table Web 1;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table Web 2;
\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table Web 3;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Balloon Text;\lsdpriority59 \lsdlocked0 Table Grid;\lsdsemihidden1 \lsdunhideused1 \lsdlocked0 Table Theme;\lsdsemihidden1 \lsdlocked0 Placeholder Text;
\lsdqformat1 \lsdpriority1 \lsdlocked0 No Spacing;\lsdpriority60 \lsdlocked0 Light Shading;\lsdpriority61 \lsdlocked0 Light List;\lsdpriority62 \lsdlocked0 Light Grid;\lsdpriority63 \lsdlocked0 Medium Shading 1;\lsdpriority64 \lsdlocked0 Medium Shading 2;
\lsdpriority65 \lsdlocked0 Medium List 1;\lsdpriority66 \lsdlocked0 Medium List 2;\lsdpriority67 \lsdlocked0 Medium Grid 1;\lsdpriority68 \lsdlocked0 Medium Grid 2;\lsdpriority69 \lsdlocked0 Medium Grid 3;\lsdpriority70 \lsdlocked0 Dark List;
\lsdpriority71 \lsdlocked0 Colorful Shading;\lsdpriority72 \lsdlocked0 Colorful List;\lsdpriority73 \lsdlocked0 Colorful Grid;\lsdpriority60 \lsdlocked0 Light Shading Accent 1;\lsdpriority61 \lsdlocked0 Light List Accent 1;
\lsdpriority62 \lsdlocked0 Light Grid Accent 1;\lsdpriority63 \lsdlocked0 Medium Shading 1 Accent 1;\lsdpriority64 \lsdlocked0 Medium Shading 2 Accent 1;\lsdpriority65 \lsdlocked0 Medium List 1 Accent 1;\lsdsemihidden1 \lsdlocked0 Revision;
\lsdqformat1 \lsdpriority34 \lsdlocked0 List Paragraph;\lsdqformat1 \lsdpriority29 \lsdlocked0 Quote;\lsdqformat1 \lsdpriority30 \lsdlocked0 Intense Quote;\lsdpriority66 \lsdlocked0 Medium List 2 Accent 1;\lsdpriority67 \lsdlocked0 Medium Grid 1 Accent 1;
\lsdpriority68 \lsdlocked0 Medium Grid 2 Accent 1;\lsdpriority69 \lsdlocked0 Medium Grid 3 Accent 1;\lsdpriority70 \lsdlocked0 Dark List Accent 1;\lsdpriority71 \lsdlocked0 Colorful Shading Accent 1;\lsdpriority72 \lsdlocked0 Colorful List Accent 1;
\lsdpriority73 \lsdlocked0 Colorful Grid Accent 1;\lsdpriority60 \lsdlocked0 Light Shading Accent 2;\lsdpriority61 \lsdlocked0 Light List Accent 2;\lsdpriority62 \lsdlocked0 Light Grid Accent 2;\lsdpriority63 \lsdlocked0 Medium Shading 1 Accent 2;
\lsdpriority64 \lsdlocked0 Medium Shading 2 Accent 2;\lsdpriority65 \lsdlocked0 Medium List 1 Accent 2;\lsdpriority66 \lsdlocked0 Medium List 2 Accent 2;\lsdpriority67 \lsdlocked0 Medium Grid 1 Accent 2;\lsdpriority68 \lsdlocked0 Medium Grid 2 Accent 2;
\lsdpriority69 \lsdlocked0 Medium Grid 3 Accent 2;\lsdpriority70 \lsdlocked0 Dark List Accent 2;\lsdpriority71 \lsdlocked0 Colorful Shading Accent 2;\lsdpriority72 \lsdlocked0 Colorful List Accent 2;\lsdpriority73 \lsdlocked0 Colorful Grid Accent 2;
\lsdpriority60 \lsdlocked0 Light Shading Accent 3;\lsdpriority61 \lsdlocked0 Light List Accent 3;\lsdpriority62 \lsdlocked0 Light Grid Accent 3;\lsdpriority63 \lsdlocked0 Medium Shading 1 Accent 3;\lsdpriority64 \lsdlocked0 Medium Shading 2 Accent 3;
\lsdpriority65 \lsdlocked0 Medium List 1 Accent 3;\lsdpriority66 \lsdlocked0 Medium List 2 Accent 3;\lsdpriority67 \lsdlocked0 Medium Grid 1 Accent 3;\lsdpriority68 \lsdlocked0 Medium Grid 2 Accent 3;\lsdpriority69 \lsdlocked0 Medium Grid 3 Accent 3;
\lsdpriority70 \lsdlocked0 Dark List Accent 3;\lsdpriority71 \lsdlocked0 Colorful Shading Accent 3;\lsdpriority72 \lsdlocked0 Colorful List Accent 3;\lsdpriority73 \lsdlocked0 Colorful Grid Accent 3;\lsdpriority60 \lsdlocked0 Light Shading Accent 4;
\lsdpriority61 \lsdlocked0 Light List Accent 4;\lsdpriority62 \lsdlocked0 Light Grid Accent 4;\lsdpriority63 \lsdlocked0 Medium Shading 1 Accent 4;\lsdpriority64 \lsdlocked0 Medium Shading 2 Accent 4;\lsdpriority65 \lsdlocked0 Medium List 1 Accent 4;
\lsdpriority66 \lsdlocked0 Medium List 2 Accent 4;\lsdpriority67 \lsdlocked0 Medium Grid 1 Accent 4;\lsdpriority68 \lsdlocked0 Medium Grid 2 Accent 4;\lsdpriority69 \lsdlocked0 Medium Grid 3 Accent 4;\lsdpriority70 \lsdlocked0 Dark List Accent 4;
\lsdpriority71 \lsdlocked0 Colorful Shading Accent 4;\lsdpriority72 \lsdlocked0 Colorful List Accent 4;\lsdpriority73 \lsdlocked0 Colorful Grid Accent 4;\lsdpriority60 \lsdlocked0 Light Shading Accent 5;\lsdpriority61 \lsdlocked0 Light List Accent 5;
\lsdpriority62 \lsdlocked0 Light Grid Accent 5;\lsdpriority63 \lsdlocked0 Medium Shading 1 Accent 5;\lsdpriority64 \lsdlocked0 Medium Shading 2 Accent 5;\lsdpriority65 \lsdlocked0 Medium List 1 Accent 5;\lsdpriority66 \lsdlocked0 Medium List 2 Accent 5;
\lsdpriority67 \lsdlocked0 Medium Grid 1 Accent 5;\lsdpriority68 \lsdlocked0 Medium Grid 2 Accent 5;\lsdpriority69 \lsdlocked0 Medium Grid 3 Accent 5;\lsdpriority70 \lsdlocked0 Dark List Accent 5;\lsdpriority71 \lsdlocked0 Colorful Shading Accent 5;
\lsdpriority72 \lsdlocked0 Colorful List Accent 5;\lsdpriority73 \lsdlocked0 Colorful Grid Accent 5;\lsdpriority60 \lsdlocked0 Light Shading Accent 6;\lsdpriority61 \lsdlocked0 Light List Accent 6;\lsdpriority62 \lsdlocked0 Light Grid Accent 6;
\lsdpriority63 \lsdlocked0 Medium Shading 1 Accent 6;\lsdpriority64 \lsdlocked0 Medium Shading 2 Accent 6;\lsdpriority65 \lsdlocked0 Medium List 1 Accent 6;\lsdpriority66 \lsdlocked0 Medium List 2 Accent 6;
\lsdpriority67 \lsdlocked0 Medium Grid 1 Accent 6;\lsdpriority68 \lsdlocked0 Medium Grid 2 Accent 6;\lsdpriority69 \lsdlocked0 Medium Grid 3 Accent 6;\lsdpriority70 \lsdlocked0 Dark List Accent 6;\lsdpriority71 \lsdlocked0 Colorful Shading Accent 6;
\lsdpriority72 \lsdlocked0 Colorful List Accent 6;\lsdpriority73 \lsdlocked0 Colorful Grid Accent 6;\lsdqformat1 \lsdpriority19 \lsdlocked0 Subtle Emphasis;\lsdqformat1 \lsdpriority21 \lsdlocked0 Intense Emphasis;
\lsdqformat1 \lsdpriority31 \lsdlocked0 Subtle Reference;\lsdqformat1 \lsdpriority32 \lsdlocked0 Intense Reference;\lsdqformat1 \lsdpriority33 \lsdlocked0 Book Title;\lsdsemihidden1 \lsdunhideused1 \lsdpriority37 \lsdlocked0 Bibliography;
\lsdsemihidden1 \lsdunhideused1 \lsdqformat1 \lsdpriority39 \lsdlocked0 TOC Heading;\lsdpriority41 \lsdlocked0 Plain Table 1;\lsdpriority42 \lsdlocked0 Plain Table 2;\lsdpriority43 \lsdlocked0 Plain Table 3;\lsdpriority44 \lsdlocked0 Plain Table 4;
\lsdpriority45 \lsdlocked0 Plain Table 5;\lsdpriority40 \lsdlocked0 Grid Table Light;\lsdpriority46 \lsdlocked0 Grid Table 1 Light;\lsdpriority47 \lsdlocked0 Grid Table 2;\lsdpriority48 \lsdlocked0 Grid Table 3;\lsdpriority49 \lsdlocked0 Grid Table 4;
\lsdpriority50 \lsdlocked0 Grid Table 5 Dark;\lsdpriority51 \lsdlocked0 Grid Table 6 Colorful;\lsdpriority52 \lsdlocked0 Grid Table 7 Colorful;\lsdpriority46 \lsdlocked0 Grid Table 1 Light Accent 1;\lsdpriority47 \lsdlocked0 Grid Table 2 Accent 1;
\lsdpriority48 \lsdlocked0 Grid Table 3 Accent 1;\lsdpriority49 \lsdlocked0 Grid Table 4 Accent 1;\lsdpriority50 \lsdlocked0 Grid Table 5 Dark Accent 1;\lsdpriority51 \lsdlocked0 Grid Table 6 Colorful Accent 1;
\lsdpriority52 \lsdlocked0 Grid Table 7 Colorful Accent 1;\lsdpriority46 \lsdlocked0 Grid Table 1 Light Accent 2;\lsdpriority47 \lsdlocked0 Grid Table 2 Accent 2;\lsdpriority48 \lsdlocked0 Grid Table 3 Accent 2;
\lsdpriority49 \lsdlocked0 Grid Table 4 Accent 2;\lsdpriority50 \lsdlocked0 Grid Table 5 Dark Accent 2;\lsdpriority51 \lsdlocked0 Grid Table 6 Colorful Accent 2;\lsdpriority52 \lsdlocked0 Grid Table 7 Colorful Accent 2;
\lsdpriority46 \lsdlocked0 Grid Table 1 Light Accent 3;\lsdpriority47 \lsdlocked0 Grid Table 2 Accent 3;\lsdpriority48 \lsdlocked0 Grid Table 3 Accent 3;\lsdpriority49 \lsdlocked0 Grid Table 4 Accent 3;
\lsdpriority50 \lsdlocked0 Grid Table 5 Dark Accent 3;\lsdpriority51 \lsdlocked0 Grid Table 6 Colorful Accent 3;\lsdpriority52 \lsdlocked0 Grid Table 7 Colorful Accent 3;\lsdpriority46 \lsdlocked0 Grid Table 1 Light Accent 4;
\lsdpriority47 \lsdlocked0 Grid Table 2 Accent 4;\lsdpriority48 \lsdlocked0 Grid Table 3 Accent 4;\lsdpriority49 \lsdlocked0 Grid Table 4 Accent 4;\lsdpriority50 \lsdlocked0 Grid Table 5 Dark Accent 4;
\lsdpriority51 \lsdlocked0 Grid Table 6 Colorful Accent 4;\lsdpriority52 \lsdlocked0 Grid Table 7 Colorful Accent 4;\lsdpriority46 \lsdlocked0 Grid Table 1 Light Accent 5;\lsdpriority47 \lsdlocked0 Grid Table 2 Accent 5;
\lsdpriority48 \lsdlocked0 Grid Table 3 Accent 5;\lsdpriority49 \lsdlocked0 Grid Table 4 Accent 5;\lsdpriority50 \lsdlocked0 Grid Table 5 Dark Accent 5;\lsdpriority51 \lsdlocked0 Grid Table 6 Colorful Accent 5;
\lsdpriority52 \lsdlocked0 Grid Table 7 Colorful Accent 5;\lsdpriority46 \lsdlocked0 Grid Table 1 Light Accent 6;\lsdpriority47 \lsdlocked0 Grid Table 2 Accent 6;\lsdpriority48 \lsdlocked0 Grid Table 3 Accent 6;
\lsdpriority49 \lsdlocked0 Grid Table 4 Accent 6;\lsdpriority50 \lsdlocked0 Grid Table 5 Dark Accent 6;\lsdpriority51 \lsdlocked0 Grid Table 6 Colorful Accent 6;\lsdpriority52 \lsdlocked0 Grid Table 7 Colorful Accent 6;
\lsdpriority46 \lsdlocked0 List Table 1 Light;\lsdpriority47 \lsdlocked0 List Table 2;\lsdpriority48 \lsdlocked0 List Table 3;\lsdpriority49 \lsdlocked0 List Table 4;\lsdpriority50 \lsdlocked0 List Table 5 Dark;
\lsdpriority51 \lsdlocked0 List Table 6 Colorful;\lsdpriority52 \lsdlocked0 List Table 7 Colorful;\lsdpriority46 \lsdlocked0 List Table 1 Light Accent 1;\lsdpriority47 \lsdlocked0 List Table 2 Accent 1;\lsdpriority48 \lsdlocked0 List Table 3 Accent 1;
\lsdpriority49 \lsdlocked0 List Table 4 Accent 1;\lsdpriority50 \lsdlocked0 List Table 5 Dark Accent 1;\lsdpriority51 \lsdlocked0 List Table 6 Colorful Accent 1;\lsdpriority52 \lsdlocked0 List Table 7 Colorful Accent 1;
\lsdpriority46 \lsdlocked0 List Table 1 Light Accent 2;\lsdpriority47 \lsdlocked0 List Table 2 Accent 2;\lsdpriority48 \lsdlocked0 List Table 3 Accent 2;\lsdpriority49 \lsdlocked0 List Table 4 Accent 2;
\lsdpriority50 \lsdlocked0 List Table 5 Dark Accent 2;\lsdpriority51 \lsdlocked0 List Table 6 Colorful Accent 2;\lsdpriority52 \lsdlocked0 List Table 7 Colorful Accent 2;\lsdpriority46 \lsdlocked0 List Table 1 Light Accent 3;
\lsdpriority47 \lsdlocked0 List Table 2 Accent 3;\lsdpriority48 \lsdlocked0 List Table 3 Accent 3;\lsdpriority49 \lsdlocked0 List Table 4 Accent 3;\lsdpriority50 \lsdlocked0 List Table 5 Dark Accent 3;
\lsdpriority51 \lsdlocked0 List Table 6 Colorful Accent 3;\lsdpriority52 \lsdlocked0 List Table 7 Colorful Accent 3;\lsdpriority46 \lsdlocked0 List Table 1 Light Accent 4;\lsdpriority47 \lsdlocked0 List Table 2 Accent 4;
\lsdpriority48 \lsdlocked0 List Table 3 Accent 4;\lsdpriority49 \lsdlocked0 List Table 4 Accent 4;\lsdpriority50 \lsdlocked0 List Table 5 Dark Accent 4;\lsdpriority51 \lsdlocked0 List Table 6 Colorful Accent 4;
\lsdpriority52 \lsdlocked0 List Table 7 Colorful Accent 4;\lsdpriority46 \lsdlocked0 List Table 1 Light Accent 5;\lsdpriority47 \lsdlocked0 List Table 2 Accent 5;\lsdpriority48 \lsdlocked0 List Table 3 Accent 5;
\lsdpriority49 \lsdlocked0 List Table 4 Accent 5;\lsdpriority50 \lsdlocked0 List Table 5 Dark Accent 5;\lsdpriority51 \lsdlocked0 List Table 6 Colorful Accent 5;\lsdpriority52 \lsdlocked0 List Table 7 Colorful Accent 5;
\lsdpriority46 \lsdlocked0 List Table 1 Light Accent 6;\lsdpriority47 \lsdlocked0 List Table 2 Accent 6;\lsdpriority48 \lsdlocked0 List Table 3 Accent 6;\lsdpriority49 \lsdlocked0 List Table 4 Accent 6;
\lsdpriority50 \lsdlocked0 List Table 5 Dark Accent 6;\lsdpriority51 \lsdlocked0 List Table 6 Colorful Accent 6;\lsdpriority52 \lsdlocked0 List Table 7 Colorful Accent 6;}}{\*\datastore 010500000200000018000000
4d73786d6c322e534158584d4c5265616465722e362e3000000000000000000000060000
d0cf11e0a1b11ae1000000000000000000000000000000003e000300feff090006000000000000000000000001000000010000000000000000100000feffffff00000000feffffff0000000000000000ffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffff
ffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffff
ffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffff
ffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffff
fffffffffffffffffdfffffffeffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffff
ffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffff
ffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffff
ffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffffff
ffffffffffffffffffffffffffffffff52006f006f007400200045006e00740072007900000000000000000000000000000000000000000000000000000000000000000000000000000000000000000016000500ffffffffffffffffffffffff0c6ad98892f1d411a65f0040963251e50000000000000000000000009030
52499eabd001feffffff00000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000ffffffffffffffffffffffff00000000000000000000000000000000000000000000000000000000
00000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000ffffffffffffffffffffffff0000000000000000000000000000000000000000000000000000
000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000ffffffffffffffffffffffff000000000000000000000000000000000000000000000000
0000000000000000000000000000000000000000000000000105000000000000}}