RT-PCR Problems with Reverse Transcription Step

Wilson Clements kendrick at u.washington.edu
Fri Sep 8 18:11:32 EST 2000

I am having problems with reproducibility in generating cDNA from RNA.  I
get variable amounts of cDNA from the same pool of isolated RNA.  I don't
think the problem is in the PCR step, because wants I have made a pool of
cDNA, I get very reproducible PCR.  Does anyone have suggestions for
increasing the reproducibility of the RT step?

So far I have tried:
1) Increasing the dilution of the RNA to decrease pipetting error.
2) Using two different RTs (Superscript 2 and Stratascript).
3) Playing with concentration of Oligo dT 18 primer (200-600ug).
4) Including or not including DTT at 10mM final.

Any suggestions will be greatly appreciated.

Wilson Clements                   e-mail:  kendrick at u.washington.edu
Department of Biochemistry
Box 357350
University of Washington.
Seattle, WA 98195

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