质粒上扩增DNA的PCR条件

质粒上扩增DNA的PCR条件

1、PCR reaction system

25 ng linear template （~6.5 kb）

50 pmol each primer

100 pmol each dNTP

1X Promega Taq buffer （no Mg2+）

1.5 mM MgCl2

1 U Taq DNA polymerase in 50 ul final volume

2、PCR programme

92°C / 2'

92°C / 30"

50°C or 55°C （depends on Tm of oligos） /30"

72°C / about 2' per kb

Go to 2, 15 times

70°C/ 8'

4°C,hold.

Takes about 2 hours to complete.

3、Notes:

If you are using Pfu turbo, use its buffer （Mg already added） and decrease elongation to 1'/kb DNA. Note that this DNA is blunt ended and can be cloned directly （no purification necessary） into a phosphorylated vector. Taq made DNA needs to be AT vector cloned