non specific bands of RT-PCR

Dirk Seegert seegert at ita.fhg.de
Fri Dec 1 04:53:36 EST 1995


Dear Tsai,
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,
best regards

Dirk Seegert, Ph.D. (called Leo)
Fraunhofer Institute Hannover, Germany




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