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.
