non specific bands of RT-PCR
seegert at ita.fhg.de
Fri Dec 1 04:53:36 EST 1995
I would try to use my specific antisense primer for cDNA synthesis first.
By using random primer you'll get a lot of unwanted shorter (or longer)
products which will substitute the primerbinding to your real cDNA in the
following PCR reaction.
Additionally we get much better results by using highly purified primers.
The longer the primers you use the more neccesary will be a purification.
In every synthesis step for oligos there is about one percent of wrong
product. So the amount of your wanted product decreases from step to step
in an exponential manner.
Another thing you might try is to use enzymes from other distributers. We
found big differences in our results by doing this. Now we are only using
enzymes from Gibco/BRL (Superscript and Taq-Pol.)
I hope this will help you,
Dirk Seegert, Ph.D. (called Leo)
Fraunhofer Institute Hannover, Germany
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