PCR reamplification problem

Z. Zhao zxz4 at PSU.EDU
Fri Jun 20 14:14:06 EST 1997


> From: edd at okstate.edu
> 
> Subject: PCR reamplification problem
> 
> Date: Fri, 20 Jun 1997 11:08:53 CST
> 
> I have had consistant problems reamplifying a PCR-generated product
> either from a dilution of the PCR mixture (I've tried a range from 1:1
> to
> 1:1E9) or from the same dilutions of a gel purified product (Qiagen,
> freeze squeeze, or remelted low-melting temp agarose). Both templates
> in
> the original PCR are from plasmid preps of a clone and the products
> are very
> specific (1700 bp) and the bands are intense. The only success has
> been
> with dilutions of an excised band from the remelted low-melting temp
> agarose but even this is very inefficient. Low dilutions of all other
> reamplifications results in a high molecular weight smear while the
> higher dilutions generally result in low molecular weight smear.
> If I use nested primers all of the above dilutions work fine with the
> exception of the Qiagen purified bands. Additionally, I have taken
> great
> care to avoid exposure to any UV light by running duplicate lanes
> which
> are aligned with the unstained lanes for excision. I know there are
> alternative approaches but I'm wondering why there should be problems
> with the reamplification. Any suggestions would be greatly
> appreciated.
> 
> Ed
> edd at okstate.edu
> ----------------------------------------------------------------------

It seems that your first round PCR generated many non-specific products 
which gave you smears in the second round. The successful PCRs with 
gel-purified templates and with nested primers seem to implicate this 
possibily. As for the phenomena that low dilutions gave high molecular 
weight smear, while higher dilutions did the opposite, I have no idea.
Well, using nested primers may be a better way to go.



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