Polymerase Chain Reaction (PCR)

Volumes used for the PCR:

Reagent Standard volume (µl)   x 4.1 (µl)
10x PCR buffer:  2.50   10.25
Mg2+ (50mM stock):  0.75    3.08
dNTPs (5mM stock):  1.00    4.10
Taq DNA polymerase (1 unit/µl):  0.50   2.05
DEPC water: 12.75    52.28
F primer (10µM):   1.25    5.13
R primer (10µM):   1.25    5.13
Total:   20      82.00

 

Conditions used for the PCR:

1) Initial denaturation at 94°C for 3 minutes

2) 94°C for 45 seconds

3) 58°C for 30 seconds                                         Step 2 to 4, 35 cycles

4) 72°C for 30 seconds

5) 72°C for 10 minutes (final extension)

6) 4°C pause

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