Long-amplicons and Taq

chovek69 via methods%40net.bio.net (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.
>
> >http://www.whitelabs.org/Lab%20Protocols/PCR%20Protocols/kyle.htm
>
> Just curious: why 65C? 5 min total extension for 3 kbp or per every kbp?
>
> Thanks,
>
> DK

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
http://www.qiagen.com/literature/qiagennews/0398/983pcro.pdf
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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