5 ´-RACE

Quicksilver 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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