PCR problem - disappearing bands
pd at mole.bio.cam.ac.uk
Thu Apr 4 05:13:22 EST 1996
In article j7r at harbinger.cc.monash.edu.au, Zhonglin Chai <zchai at cobra.path.monash.edu.au > writes:
>kunazhe3 at caos.kun.nl (KUN Hematol.Lab.) wrote:
>>We have, as so many, a puzzling problem with our PCR results. When we
>>perform a specific PCR reaction we get reproducibly three bands, about 400
>>bp, 475 bp and 550 bp. The first two are the expected bands (alternative
>>splicing) the third band might be another alternative spliced form or an
>>aspecific product. We use primers flanking the alternative spliced exon
>>(400 and 475 bp). When we perform under the same conditions with the
>>product of the first reaction as a source of DNA (the mixture) and the
>>same primer set another PCR reaction we loose the band of 475 bp. Why are
>>not the same bands amplifyed in the second reaction which are
>>amplifyed in the first reaction? What are we missing, could anyone
>>clarify to us why we are loosing the 475 bp band in the second reaction?
>Can you re-amplify the three single banded products by cutting the bands into
>the next PCR Rxs, under the same conditions?
This reminds me of something that happened to me once.
A set of primers I was trying out gave me three bands. In order to find out
which was the real band, I cut out the largest band, and re-PCR'd it using the
same primers and conditions. All I got was the other two bands! No sign of the
large band at all.
Never did work out what was going on...
[E-mail pd at mole.bio.cam.ac.uk]
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