PCR amplification problems could use some help

Francesca Catalano fcatala at luc.edu
Tue Dec 7 01:58:49 EST 1999


I have done PCR to get much larger pieces... what about your DNA?  Maybe
it is not a good prep?  Do you use an oil overlay?  I have heard that
old mineral oil (not protected from light) can break down and inhibit
PCR.  Are you really confident in the primers?  Do they work in
different sets.. i.e., does the 3' primer work with another 5' primer..
not the one of interest here?  Hmm I have other ideas.. let me know..

:-)
Francesca Catalano
fcatala at luc.edu

Scott C Winkel wrote:
> 
> I am working on an amplification of a gene that is 1109 bp. This particular
> assay has worked fine in other labs but has yet to be successful within our
> facility. Other assays of smaller base pair length are working great, but
> for some unknown reason this larger size assay does not want to cooperate.
> Our primers have been checked and seem to be working fine. Actually , all we
> seem to be getting are primer dimers. We are using new dNTP's, taq +Ab,
> Mg++, & 10x buffer. The protocol is identical to what has worked flawlessly
> in other labs. The only variables that are different are the technician,
> location, water, and machine. We are currently using the Perkins Elmer 9700
> thermocycler.
> 
> If anyone has experience amplifying fragments this large I would love to
> discuss the methods by which they are using.
> 
> Thanks
> 
> Scott
> 
> scwinkel at prodigy.net



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