Need help with Long PCR
thomas at molbio.uoregon.edu
Mon Apr 17 02:28:20 EST 1995
Dear Sam, Check out a recent short article in TIBS by WM Barnes (aka Mr
long PCR) on essential elements of the technique. Also S Chengs recent
Nature article. Barnes seems to think a Robocycler (Stratagene) is
essential, but a capillary PCR like Idaho Tech would also work if faster
ramp times are important. Barnes also really prefers the KlenTaq 1/ Pfu
combination, but he invented KlenTaq 1... Good Luck
Institute of Molecular Biology,
University of Oregon
In article <3mrmcc$1fo0 at news.doit.wisc.edu>, shender at macc.wisc.edu (Sam
> Dear Netters
> I'm sure this topic has been beat to death in this
> newsgroup and I'm sorry to bring it up again. Has anyone
> written a FAQ on this topic?
> I have been trying to amplify an 11 kb fragment from a
> plasmid using Stratagenes Taq extender. My primers are both
> 33 nts each. I do the following PCR program.
> Segment 1 94oC 10sec
> Segment 2 56oC 1min
> Segment 3 72oC 10min with auto extension of 10sec/cycle.
> Cylces 25
> Taq 15units
> taq ext. 15units
> I have tried varing some of the parameters, as follows: I dropped the
> extension temp down to 68oC, I used different dNTP's (Promega and
> Pharmacia), varied the amount of template from 100-500ng. None of these
> seemed to help much.
> Using different primers the largest fragment I have been able to
> amplify is 6kb. Everything larger than that, and most times all my
> reactions, run as a big smear on an ethidium stained gel. I also often
> see bright staining trapped in the wells. Can anyone give me some
> general guidelines that
> work well for long PCR? I am basically trying to amplify the entire
> circular plasmid, could this be the problem? Any help would be
> and can either be posted or sent to me via email to
> shender at macc.wisc.edu.
> Sam Henderson
> Shender at macc.wisc.edu
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