"BEST" PCR—从质粒上扩增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.