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PCR ghost bands: an update

Robert Preston rapr at MED.PITT.EDU
Fri Jul 1 14:29:01 EST 1994

> Briefly, all attempts failed to yeield a solution:
> - The quality of a few particularly "bad primers" was tested by acrylamide
>   gel electrophoresis of the 32P-kinased primers. They appear to be 
>   essentially pure, probably 99+% (eyeballed).
> - Amplification was done with two pairs of primers in the same reaction. 
>   The "good" pair gave one band, the "bad" pair gave both its correct and
>   incorrect bands.
> Pierre

How about testing those bad primers by a method that would detect a mixture
of primers where some fraction has the wrong base at one or more points.
It seems possible that one or more of your oligo synthesis facilities 
is inadvertently synthesizing partially "degenerate" oligos, either by
operator or machine malfunction, or deliberate sabotage by a disgruntled
employee.  I don't know the best way to do that, whether HPLC or possibly
nondenaturing PAGE (a la SSCP), or complete hydrolysis and quantitative
analysis of nucleotides?

Rob Preston
rapr at med.pitt.edu

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