BEST" PCR

BEST" PCR conditions for amplifying DNA from plasmids

25 ng linear template (~6.5 kb)
50 pmol each primer
100 pmol each dNTP
1X Promega Taq buffer (no Mg++)
1.5 mM MgCl₂
1 U Taq DNA polymerase in 50 ul final

1. 92°C / 2'
2. 92°C / 30"
3. 50°C or 55°C (depends on Tm of oligos) /30"
4. 72°C / about 2' per kb
5. Go to 2, 15 times
6. 70°C/ 8'
7. 4°C...hold.

Takes about 2 hours to complete.

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.

For PCR from yeast genomic DNA , good DNA is important. Grow cells in Ade (red pigment kills PCR ) and if you have trouble, you can gene clean the DNA .

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