PCR Master Mix

50 ㎕

Template DNA

10~100 ng

primer 1 (100 pmole)

1~2 ㎕

primer 2 (100 pmole)

1~2 ㎕

Sterilized deionized water

Up to 100 ㎕

95°C

3 min

1 cycle

94°C

30 sec

30 cycle

Annealing*

30 sec

72°C

~2 min*

72°C

5 min

1 cycle

*Annealing temperature : Tm - (~ 5 °C) / Tm : 2 X (A+T) + 4 X (G + C)

*Extension time
~ 2 kb : 1 min
> 2 kb : 1 min + (2.5 sec / 100 bases)
If PCR fragments are to be cloned into T/A vectors, the last extension step can be extended to up to 30 min.