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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