| 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
- 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.
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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