Long-amplicons and Taq
(by ivanoov from gmail.com)
Sun Jul 10 09:28:02 EST 2011
On Jul 8, 3:16 pm, d... from noemail.thankstospam.net (DK) wrote:
> In article <740568f1-764d-4381-aa90-15b203e79... from x41g2000yqd.googlegroups.com>, chovek69 <ivan... from gmail.com> wrote:
> >I regularly amplify >3kb with regular Taq with (NH4)2SO4 buffer (see
> >link below) + 1M Betaine and 5min extension step at 65C.
> Just curious: why 65C? 5 min total extension for 3 kbp or per every kbp?
It is 65C for 5min total ext time and betaine is critical.
There are some reports stating that decreasing extension and
denaturation temp could improve long PCR. See for example this
although there many others.
I think it is because of the damage to the template during the longer
cycles plus betaine could decrease the Tm a degree or two. Of course
the the amplicon %GC is most important for the optimal ext temp so
it is best to run a gradient optimization.
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