Need help with Long PCR

Matthew Thomas 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
                                                Ted Michelini
                                                Institute of Molecular Biology,
                                                University of Oregon




In article <3mrmcc$1fo0 at news.doit.wisc.edu>, shender at macc.wisc.edu (Sam
Henderson) wrote:

> 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
> appreciated
> and can either be posted or sent to me via email to
> shender at macc.wisc.edu.
> Thanks
> Sam Henderson
> Shender at macc.wisc.edu



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