RACE

Cyrill Steiner cyrill.steiner at ipw.biol.ethz.ch
Fri Jul 9 11:22:50 EST 1999


Hello,

who has a lot of RACE experience ?
I tried the Boeringer-RACE Kit for a 5' RACE. I followed exactly the
kits protocol but I failed to amplify anything specific. Controls
indicate that the cDNA-Synthesis is working but that there is a trouble
with the  terminal transferase.

How can I increase the amount of  cDNA poduced and how to inrease the
efficiency of the terminal transferase?

Is more RNA, more enzyme and more primer a good recipie to increase cDNA
synthesis?
How do I increase the efficiency of the terminal transferase reaction ?

So far I tested:
-mRNA instead of total RNA
-reverse Transcription at 37 and 42 °C (instead of 55 as indicated in
the kit)
-other reverse transcriptases (Superscript II, Stratragene) than the one
of the boeringer kit
-PCR annealing temperatures at 55, 50 and 45 °C

Has anybody made better expriences by ligating oligo-primers to the cDNA
instead of using terminal transferase?

Thanks a lot for any answers

Cyrill




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