Quantitative PCR (qPCR) and "tripple bands"

Michael Morales mmorales at acpub.duke.edu
Thu Jun 15 10:59:03 EST 1995

Holger Bohnemeier (Bohnemeier at ukbf.fu-berlin.de) wrote:
: In order to quantitate mRNA from several tissues I've established an
: RT-qPCR. I'm dealing with a 450 bp wildtype amplicon and a 410 bp dele-
: tion mutante. If either one of the above is shown on an EtBr-stained
: agarose gel all seems rather fine. If both are put in a single PCR
: together (as it is usual for qPCR) sometimes I can see a so called
: "tripple band" instead of the expected double band (the signal of the
: wildtype and the deletion-mutante). This extra band is just between
: the to expected one. I guess it is a double stranded hybrid synthesized
: out of a wildtype and a deletion-strand.

: The question to the community now: Has anyone notified such an artefact
: to?

I have.  I never really figured out what it was, but it seemed to disappear
when I ran the reaction products on a nondenaturing polyacrylamide/TBE gel
instead of my normal agarose/TAE gel.  Hope this helps.

Mike Morales

: Any suggestions or ideas are welcome!
: Bye!

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