Long-amplicons and Taq

Cathal Garvey via methods%40net.bio.net (by cathalgarvey from gmail.com)
Sun Jul 10 11:48:50 EST 2011


Thanks to everyone for the responses. That Qiagen article was surprisingly
helpful, though it would be nice for them to share the constitution of their
"Q Solution".

In particular I was interested to read about the ammonium ion effects and
the effects of high temperatures; the high-temperature depurination issue
may have been behind some past PCR failures of mine. Good to know!

Thanks again,
Cathal

On 10 July 2011 15:28, chovek69 <ivanoov from gmail.com> wrote:

> 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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