PCR problem - disappearing bands

Zhonglin Chai zchai at cobra.path.monash.edu.au
Wed Apr 3 20:23:05 EST 1996


kunazhe3 at caos.kun.nl (KUN Hematol.Lab.) wrote:
>Dear world,
>
>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?






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