Bitan:PCR Amplification of the Initial ssDNA Library for SELEX experiments

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PCR Amplification of the Initial Library for SELEX<o:p></o:p>

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Template DNA containing the randomized sequence (A:T:G:C=1:1:1:1) was obtained from Dr. Betty Chen (Department of Biological Chemistry).  It is not purified.  The absorbance at A260 of DNA is 1.010 (in a 100-fold diluted sample), which is equivalent to 0.97 μM.  Thus, the concentration of template DNA solution is 97 μM.<o:p></o:p>

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Prepare PCR mix as shown below. If more than one PCR reaction is performed, prepare a master mixture).  PCR cycle is programmed into the PCR machine under the name “APTKAZU” or “APTAMER”.<o:p></o:p>

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Water                        96 μl<o:p></o:p>

Template DNA          5 μl (0.49 nmole, BC-SELEX-TEMP2)<o:p></o:p>

10× Taq buffer         20 μl <o:p></o:p>

25 mM MgCl2          60 μl<o:p></o:p>

10 mM dNTPs          14 μl<o:p></o:p>

Primer forward         2 μl (BC-SELEC-F)<o:p></o:p>

Primer reverse          2 μl (BC-SELEX-R)<o:p></o:p>

Taq polymerase        1 μl <o:p></o:p>

TOTAL                     200 ml<o:p></o:p>

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“APTKAZU” program<o:p></o:p>

Denature 94 °C, 5 min. (without hot start)<o:p></o:p>

<![if !vml]><img width=16 height=56 src="PCR%20for%20initial%20library_files/image001.png" v:shapes="_x0000_s1029"><![endif]>Denature 94 °C, 30 sec.<o:p></o:p>

Anneal 52 °C, 30 sec.          30 cycles<o:p></o:p>

Extend 72 °C, 30 sec.<o:p></o:p>

Extend 72 °C, 7 min.<o:p></o:p>

Storage 10 °C<o:p></o:p>

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“APTAMER” program<o:p></o:p>

Denature 94 °C, 5 min. (without hot start)<o:p></o:p>

<![if !vml]><img width=16 height=56 src="PCR%20for%20initial%20library_files/image002.png" v:shapes="_x0000_s1030"><![endif]>Denature 94 °C, 30 sec.<o:p></o:p>

Anneal 52 °C, 30 sec.          20 cycles<o:p></o:p>

Extend 72 °C, 30 sec.<o:p></o:p>

Extend 72 °C, 7 min.<o:p></o:p>

Storage 10 °C<o:p></o:p>

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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>

Measure the DNA concentration. <o:p></o:p>

<a href="http://openwetware.org/wiki/Bitan:todo">Back to To-Do List</a><o:p></o:p>

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