Bitan:PCR Amplification of the Initial ssDNA Library for SELEX

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<head> <meta name=Title content="PCR Amplification of the Initial Library for SELEX"> <meta name=Keywords content=""> <meta http-equiv=Content-Type content="text/html; charset=macintosh"> <meta name=ProgId content=Word.Document> <meta name=Generator content="Microsoft Word 11"> <meta name=Originator content="Microsoft Word 11"> <link rel=File-List href="PCR%20for%20initial%20library_files/filelist.xml"> <link rel=Edit-Time-Data href="PCR%20for%20initial%20library_files/editdata.mso"> <!--[if !mso]> <style> v\:* {behavior:url(#default#VML);} o\:* {behavior:url(#default#VML);} w\:* {behavior:url(#default#VML);} .shape {behavior:url(#default#VML);} </style> <![endif]--> <title>PCR Amplification of the Initial Library for SELEX</title> <!--[if gte mso 9]><xml>

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<tr>
 <td width=443 valign=top style='width:442.8pt;border:solid windowtext .5pt;
 background:#CCFFCC;mso-shading:windowtext;mso-pattern:solid #CCFFCC;
 padding:0cm 5.4pt 0cm 5.4pt'>
 <p class=MsoNormal align=center style='text-align:center'><span
 style='font-size:14.0pt;font-family:Calibri;color:#993300'><b>PCR
 Amplification of the Initial Library for SELEX<o:p></o:p></b></span></p>
 <p class=MsoNormal><span style='font-size:14.0pt;font-family:Calibri;
 color:#993300'><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></span></p>
 <p class=MsoNormal><span style='font-size:14.0pt;font-family:Calibri;
 color:#993300'>Template DNA containing the randomized sequence
 (A:T:G:C=1:1:1:1) was obtained from Dr. Betty Chen (Department of Biological
 Chemistry).<span style="mso-spacerun: yes">&nbsp; </span>It is not
 purified.<span style="mso-spacerun: yes">&nbsp; </span>The absorbance at A<sub>260
 </sub>of DNA is 1.010 (in a 100-fold diluted sample), which is equivalent to
 0.97 &#956;M.<span style="mso-spacerun: yes">&nbsp; </span>Thus, the
 concentration of template DNA solution is 97 &#956;M.<o:p></o:p></span></p>
 <p class=MsoNormal><span style='font-size:14.0pt;font-family:Calibri;
 color:#993300'><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></span></p>
 <p class=MsoNormal style='tab-stops:108.0pt'><span style='font-size:14.0pt;
 font-family:Calibri;color:#993300'>Prepare PCR mix as shown below. If more
 than one PCR reaction is performed, prepare a master mixture).<span
 style="mso-spacerun: yes">&nbsp; </span>PCR cycle is programmed into the PCR
 machine under the name “APTKAZU” or “APTAMER”.<o:p></o:p></span></p>
 <p class=MsoNormal style='tab-stops:108.0pt'><span style='font-size:14.0pt;
 font-family:Calibri;color:#993300'><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></span></p>
 <p class=MsoNormal style='tab-stops:108.0pt'><span style='font-size:14.0pt;
 font-family:Calibri;color:#993300'>Water<span style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>96
 &#956;l<o:p></o:p></span></p>
 <p class=MsoNormal style='tab-stops:108.0pt'><span style='font-size:14.0pt;
 font-family:Calibri;color:#993300'>Template DNA<span style='mso-tab-count:
 1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span><span
 style="mso-spacerun: yes">&nbsp; </span>5 &#956;l (0.49 nmole,
 BC-SELEX-TEMP2)<o:p></o:p></span></p>
 <p class=MsoNormal style='tab-stops:108.0pt'><span style='font-size:14.0pt;
 font-family:Calibri;color:#993300'>10&times; Taq buffer<span
 style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>20
 &#956;l <o:p></o:p></span></p>
 <p class=MsoNormal style='tab-stops:108.0pt'><span style='font-size:14.0pt;
 font-family:Calibri;color:#993300'>25 mM MgCl<sub>2</sub><span
 style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>60
 &#956;l<o:p></o:p></span></p>
 <p class=MsoNormal style='tab-stops:108.0pt'><span style='font-size:14.0pt;
 font-family:Calibri;color:#993300'>10 mM dNTPs<span style='mso-tab-count:
 1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>14 &#956;l<o:p></o:p></span></p>
 <p class=MsoNormal style='tab-stops:108.0pt'><span style='font-size:14.0pt;
 font-family:Calibri;color:#993300'>Primer forward<span style='mso-tab-count:
 1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span><span style="mso-spacerun:
 yes">&nbsp; </span>2 &#956;l (BC-SELEC-F)<o:p></o:p></span></p>
 <p class=MsoNormal style='tab-stops:108.0pt'><span style='font-size:14.0pt;
 font-family:Calibri;color:#993300'>Primer reverse<span style='mso-tab-count:
 1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span><span
 style="mso-spacerun: yes">&nbsp; </span>2 &#956;l (BC-SELEX-R)<o:p></o:p></span></p>
 <p class=MsoNormal style='tab-stops:108.0pt'><span style='font-size:14.0pt;
 font-family:Calibri;color:#993300'><u>Taq polymerase<span style='mso-tab-count:
 1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span><span style="mso-spacerun:
 yes">&nbsp; </span>1 &#956;l <o:p></o:p></u></span></p>
 <p class=MsoNormal style='tab-stops:108.0pt'><span style='font-size:14.0pt;
 font-family:Calibri;color:#993300'>TOTAL<span style="mso-spacerun:
 yes">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 </span>200 ml<o:p></o:p></span></p>
 <p class=MsoNormal style='tab-stops:108.0pt'><span style='font-size:14.0pt;
 font-family:Calibri;color:#993300'><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></span></p>
 <p class=MsoNormal style='tab-stops:108.0pt'><span style='font-size:14.0pt;
 font-family:Calibri;color:#993300'>“APTKAZU” program<o:p></o:p></span></p>
 <p class=MsoNormal style='tab-stops:108.0pt'><span style='font-size:14.0pt;
 font-family:Calibri;color:#993300'>Denature 94 °C, 5 min. (without hot start)<o:p></o:p></span></p>
 <p class=MsoNormal style='tab-stops:108.0pt'><!--[if gte vml 1]><v:shapetype
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   <v:f eqn="sum #1 0 #0"/>
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   <v:f eqn="prod #0 9598 32768"/>
   <v:f eqn="sum 21600 0 @4"/>
   <v:f eqn="sum 21600 0 #1"/>
   <v:f eqn="min #1 @6"/>
   <v:f eqn="prod @7 1 2"/>
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 style='font-size:14.0pt;font-family:Calibri;color:#993300'>Denature 94 °C, 30
 sec.<o:p></o:p></span></p>
 <p class=MsoNormal style='tab-stops:108.0pt'><span style='font-size:14.0pt;
 font-family:Calibri;color:#993300'>Anneal 52 °C, 30 sec.<span
 style="mso-spacerun:
 yes">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>30 cycles<o:p></o:p></span></p>
 <p class=MsoNormal style='tab-stops:108.0pt'><span style='font-size:14.0pt;
 font-family:Calibri;color:#993300'>Extend 72<sup> </sup>°C, 30 sec.<o:p></o:p></span></p>
 <p class=MsoNormal style='tab-stops:108.0pt'><span style='font-size:14.0pt;
 font-family:Calibri;color:#993300'>Extend 72 °C, 7 min.<o:p></o:p></span></p>
 <p class=MsoNormal style='tab-stops:108.0pt'><span style='font-size:14.0pt;
 font-family:Calibri;color:#993300'>Storage 10 °C<o:p></o:p></span></p>
 <p class=MsoNormal><span style='font-size:14.0pt;font-family:Calibri;
 color:#993300'><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></span></p>
 <p class=MsoNormal><span style='font-size:14.0pt;font-family:Calibri;
 color:#993300'>“APTAMER” program<o:p></o:p></span></p>
 <p class=MsoNormal style='tab-stops:108.0pt'><span style='font-size:14.0pt;
 font-family:Calibri;color:#993300'>Denature 94 °C, 5 min. (without hot start)<o:p></o:p></span></p>
 <p class=MsoNormal style='tab-stops:108.0pt'><!--[if gte vml 1]><v:shape
  id="_x0000_s1030" type="#_x0000_t88" style='position:absolute;margin-left:132pt;
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  strokeweight="2.25pt"/><![endif]--><![if !vml]><span style='mso-ignore:vglayout;
 position:relative;z-index:1'><span style='position:absolute;left:130px;
 top:-2px;width:16px;height:56px'><img width=16 height=56
 src="PCR%20for%20initial%20library_files/image002.png" v:shapes="_x0000_s1030"></span></span><![endif]><span
 style='font-size:14.0pt;font-family:Calibri;color:#993300'>Denature 94 °C, 30
 sec.<o:p></o:p></span></p>
 <p class=MsoNormal style='tab-stops:108.0pt'><span style='font-size:14.0pt;
 font-family:Calibri;color:#993300'>Anneal 52 °C, 30 sec.<span
 style="mso-spacerun:
 yes">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>20 cycles<o:p></o:p></span></p>
 <p class=MsoNormal style='tab-stops:108.0pt'><span style='font-size:14.0pt;
 font-family:Calibri;color:#993300'>Extend 72<sup> </sup>°C, 30 sec.<o:p></o:p></span></p>
 <p class=MsoNormal style='tab-stops:108.0pt'><span style='font-size:14.0pt;
 font-family:Calibri;color:#993300'>Extend 72 °C, 7 min.<o:p></o:p></span></p>
 <p class=MsoNormal style='tab-stops:108.0pt'><span style='font-size:14.0pt;
 font-family:Calibri;color:#993300'>Storage 10 °C<o:p></o:p></span></p>
 <p class=MsoNormal style='tab-stops:108.0pt'><span style='font-size:14.0pt;
 font-family:Calibri;color:#993300'><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></span></p>
 <p class=MsoNormal style='tab-stops:108.0pt'><span style='font-size:14.0pt;
 font-family:Calibri;color:#993300'>Purify the DNA product after confirming
 product generation by gel electrophoresis. Use <a
 href="http://www1.qiagen.com/Products/DnaCleanup/GelPcrSiCleanupSystems/QIAquickPCRPurificationKit.aspx#Tabs=t1">Qiaquick
 Qiagen PCR purification kit</a> (catalogue # 28104). <o:p></o:p></span></p>
 <p class=MsoNormal style='tab-stops:108.0pt'><span style='font-size:14.0pt;
 font-family:Calibri;color:#993300'>Measure the DNA concentration. <o:p></o:p></span></p>
 <p class=MsoNormal style='tab-stops:108.0pt'><span style='font-size:14.0pt;
 font-family:Calibri;color:#993300'><a
 href="http://openwetware.org/wiki/Bitan:todo"><span style='color:#993300'>Back
 to To-Do List</span></a><b><o:p></o:p></b></span></p>
 </td>
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