PCR problem - please help

[Shahram Mori]

Vince Mulholland (mulholl at sasa.gov.uk) wrote:
: I've got a contaminating band in my PCR reactions. I'm using 
: allele-specific amplification to differentiate between species of 
: nematodes. The set-up is as follows;

:        'A'-specific         'B'-specific                    Universal
:             -->                  -->                          <--
:           390 bp               240 bp

: So, the PCR reaction contains three primers. I get a particular band 
: for each species or both bands when there is a mixture. This has been 
: working fine up until a few weeks ago. I still get a single band when 
: I amplify a single species. However, when there is a mixture of the 
: two species I get a contaminating 800 bp band. This is the odd part - 
: it is ONLY present when there is a mixture.

: Anyone had similar problems? Any ideas gratefully accepted.


: Thanks in advance, Dear Vince, Though this does apparent weird, the fact
that if the two primers are in a mixture leads me to believe that there ia
a contaminating DNA that is amplified by Primers A and B. Either the DNA
is in the primers or in the source template. I would advise to run a 5%
Acrylamide gel of the the template and a 20% gel of your primers. Gel
purify both and use in new Amplification reactions. This should get rid of
it; I hope. 
Cheers

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Shahram Mori
Program in Molecular Biology
Department of Chemistry and Biochemistry Box 3C
New Mexico State University
Las Cruces NM
88003
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