5´-RACE

[Michael Lassner]

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.
_______________________________________________________
Quicksilver wrote:
> 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.

> Roger