two first PCR cycles with long times?
MREEBEN at BIOMED.UIO.NO
Fri Jul 24 17:08:31 EST 1992
Accidentally in a recent research article in Science from 3 July 1992 Vol. 257
p.55 I came over following PCR conditions.
...All PCR were performed on single stranded cDNA's generated from
poly(A)+-enriched RNA from different brain regions.
The PCR conditions with nonredundant oligonucleotides consisted of two cycles
at 95 C (5 minutes), 62 to 68 C (2 minutes), and 72 C (10 minutes) followed by
25 cycles of 95 C (1 min), 62 to 68 C (2 minutes), and 72 C (2 minutes).
I would be grateful, if somebody can explain me the sense of these first two
cycles. OK, I understand initial more thorough denaturation, but why second
long denaturation ? And why two 10 min extensions in the beginning ? Is initial
template considered to be worse for elongation ? And how much this trick can
improve PCR ?
Besides any hints I would really appreciate some references on the theme.
Thank you in advance.
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