mikel51 at mother.com
Wed Oct 1 08:05:22 EST 1997
One trick that I have found useful is to use longer primers. Then you
can use high anneal temperatures and do touchdown PCR. I have had good
luck with 28-31 mer primers. Then do your PCR with 5 cycles of 72
anneal, 5 cycles of 70 degree anneal, and 30 cycles of 68 degree
anneal. It also may help to use a hot start PCR if you are not already
doing so. I have had good luck with the mixture polymerases, They are
predominantly Taq, but also have a small amount of proofreading enzyme.
Boehringer and Clontech sell these type of enzymes.
> 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.
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