Long-amplicons and Taq
(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
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!
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
> 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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