RT-PCR Problems with Reverse Transcription Step
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
University of Washington.
Seattle, WA 98195
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