PCR amplification problems could use some help

Chris LaRosa plarosa1 at navix.net
Sun Jan 2 21:24:11 EST 2000


1.1kb is small, not large and should present no problem for a compentent
research tech.  You need to examine the purity and quantity of the template, and
properties of the primers if things dont work.  We use Oligo 4 on a mac to
examine the primer tm, binding properties of the primers....Usually if things
dont work its easiest to redesign primers than to optimize.   If you want to try
some quick optimization , try titrating the Mg conc.    I would suggest you do a
web search using metacrawler, or mamma.com for pcr protocols... these are easily
found.   Many web sites offer trouble shooting  guides for pcr problems  .   The
companies that sell the Taq also offer tech support documents on their web sites
that go into the many details of PCR.    There really is a lot of information on
the web on this topic which you can find in about 15 minutes so i am not going
into the details.  Usually the problem is that the primer design is not the
best. If you are getting primer dimers this could because the primers bind to
each other or the template is bad.  You will always get a product with pcr,,,,,
but often not the correct one.   Without knowing more details of your problem it
s impossible to trouble shoot it.

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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