PCR problem

Arnoud van Vliet avvliet at knoware.nl
Mon Aug 21 16:40:18 EST 2000


> I used forward and reverse primers derived from conserved sequences
> available in literature to hook out a particular gene.A single
> amplified product of 1.4kb using both the primers together as well as
> using the reverse primer alone was obtained, but no amplication was
> obtained using the forward primer alone.I want to know whether it is
> possible to hook out the full gene by using the single primer or can it
> be a part of the gene or what?Thanks

When I had just started my Ph.D. project, I wanted to amplify a complete
bacterial 16S rDNA sequence (approx 1500 bp). However, I just got a 1200 bp
product. I cloned it, and found it was part of the 16S rDNA, but only
contained bp 300-1500.

When I reordered the forward primer, suddenly the 1200 bp product
disappeared and the 1500 bp fragment appeared. I deducted that the forward
primer had been degraded, and that the reverse primer had been able to do
the partial job on its own. But, as it was unlikely that I would be awarded
a Ph.D. on a thesis about PCR artifacts, I decided to put the subjects
skeleton in the closet.

Arnoud








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