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

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PCR Amplification of the Initial Library for SELEX

PCR Amplification of the Initial Library for SELEX

 

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.

 

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”.

 

Water                        96 μl

Template DNA          5 μl (0.49 nmole, BC-SELEX-TEMP2)

10× Taq buffer         20 μl

25 mM MgCl2          60 μl

10 mM dNTPs          14 μl

Primer forward         2 μl (BC-SELEC-F)

Primer reverse          2 μl (BC-SELEX-R)

Taq polymerase        1 μl

TOTAL                     200 ml

 

“APTKAZU” program

Denature 94 °C, 5 min. (without hot start)

Denature 94 °C, 30 sec.

Anneal 52 °C, 30 sec.          30 cycles

Extend 72 °C, 30 sec.

Extend 72 °C, 7 min.

Storage 10 °C

 

“APTAMER” program

Denature 94 °C, 5 min. (without hot start)

Denature 94 °C, 30 sec.

Anneal 52 °C, 30 sec.          20 cycles

Extend 72 °C, 30 sec.

Extend 72 °C, 7 min.

Storage 10 °C

 

Purify the DNA product after confirming product generation by gel electrophoresis. Use Qiaquick Qiagen PCR purification kit (catalogue # 28104).

Measure the DNA concentration.

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