PCR problem - disappearing bands

Bernard Murray bernard at elsie.nci.nih.gov
Fri Apr 5 02:35:03 EST 1996


>Well this looks as if the largest band is a heteroduplex of a longer and 
>smaller fragment with a protruding single-stranded part that retards 
>migration in gels.

Oops, I feel very guilty now as I posted a request for information on
sorting out heteroduplex problems several months ago and received
several very useful replies but did not get around to posting a summary.
I believe that most of the tips that I was given have been mentioned
by individuals but here is a belated summary;

1)	Denaturing acrylamide gel should give two bands.
2)	Increase primer concentration and/or decrease number of cycles
	and this should reduce/eliminate the mystery band.
3)	Mix the two pure "expected" bands, heat and slow anneal then
	run electrophoresis as normal.  You should get three bands again.
4)	You should get all three bands if you perform PCR on the
	mystery band.

I recommend (1) and (3) for diagnosis and (2) if this is a problem
on a routine basis.

An obvious warning is don't try and subclone the mystery band unless
it fails the above tests.
	A tardy thank you to all who replied to me last October and
my apologies to the net in general for not posting a summary.

		Bernard

Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov  (National Cancer Institute, NIH, Bethesda MD, USA)




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