Bitan:PCR Amplification of the Initial ssDNA Library for SELEX experiments
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border:none;mso-border-alt:solid windowtext .5pt;mso-padding-alt:0cm 5.4pt 0cm 5.4pt'> <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]> <![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"> </span>It is not purified.<span style="mso-spacerun: yes"> </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 μM.<span style="mso-spacerun: yes"> </span>Thus, the concentration of template DNA solution is 97 μM.<o:p></o:p></span></p> <p class=MsoNormal><span style='font-size:14.0pt;font-family:Calibri; color:#993300'><![if !supportEmptyParas]> <![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"> </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]> <![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'> </span>96 μl<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'> </span><span style="mso-spacerun: yes"> </span>5 μl (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× Taq buffer<span style='mso-tab-count:1'> </span>20 μl <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'> </span>60 μl<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'> </span>14 μl<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'> </span><span style="mso-spacerun: yes"> </span>2 μl (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'> </span><span style="mso-spacerun: yes"> </span>2 μl (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'> </span><span style="mso-spacerun: yes"> </span>1 μl <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">
</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]> <![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 id="_x0000_t88" coordsize="21600,21600" o:spt="88" adj="1800,10800" path="m0,0qx10800@0l10800@2qy21600@11,10800@3l10800@1qy0,21600e" filled="f"> <v:formulas> <v:f eqn="val #0"/> <v:f eqn="sum 21600 0 #0"/> <v:f eqn="sum #1 0 #0"/> <v:f eqn="sum #1 #0 0"/> <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"/> <v:f eqn="prod #0 2 1"/> <v:f eqn="sum 21600 0 @9"/> <v:f eqn="val #1"/> </v:formulas> <v:path arrowok="t" o:connecttype="custom" o:connectlocs="0,0;21600,@11;0,21600" textboxrect="0,@4,7637,@5"/> <v:handles> <v:h position="center,#0" yrange="0,@8"/> <v:h position="bottomRight,#1" yrange="@9,@10"/> </v:handles> </v:shapetype><v:shape id="_x0000_s1029" type="#_x0000_t88" style='position:absolute; margin-left:132pt;margin-top:2.95pt;width:9.65pt;height:50.15pt;z-index:1; mso-position-horizontal:absolute;mso-position-vertical:absolute' strokeweight="2.25pt"/><![endif]--><![if !vml]><span style='mso-ignore:vglayout; position:absolute;z-index:0;margin-left:130px;margin-top:1px;width:16px; height:56px'><img width=16 height=56 src="PCR%20for%20initial%20library_files/image001.png" v:shapes="_x0000_s1029"></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"> </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]> <![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; margin-top:.2pt;width:9.65pt;height:50.15pt;z-index:2; mso-position-horizontal:absolute;mso-position-vertical:absolute' 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"> </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]> <![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> </tr>
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