beckerr at vzdmzd.zdv.uni-mainz.de
Wed Oct 1 09:03:28 EST 1997
Hi, dear newsgroupers,
we have tremedous problems in performing 5´-RACE. We use the Gibco
protocol but use different anchor and adaptor primers. In this respect
we follow the original protocol from Frohman et al. They perform
tailing with d(A) (Gibco uses d(C)) and we use the Qo, Q1 and QT
primers (Gibco uses an Inositol containing anchor primer). In the
first round PCR we get already a high molecular weight smear, which is
strongly amplified in second round PCR. We us mRNA, digested with
DNAse. After reverse transcription we digest with RNAse. Raising the
temperature reduces the smear but it is not totally abolished.
Who has experience with this method and can help us?.
Thanks a lot for your suggestions
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