help with 5' race

[Peter J. Neame]

Eric C. Anderson (anderson at pharmdec.wustl.edu) wrote:

: i have tried both the Gibco kit and one from CLONTECH.  both of these kits

Ditto on the Clontech kit. We've used it for a couple of proteins where 
we wanted to check that the N-terminal wasn't preceded by anything other 
than a signal sequence. In both cases, we got about 200 bp of sequence 
upstream of the N-terminal. Our 3' primers were in the region 100 -200 3' 
of the N-terminal and were not significantly degenerate. 

One caveat; the 3' end of the PCR product is liable to be the best 
quality. We have only sequenced 3' -> 5' (direct sequencing of the PCR 
product) due to "fuzziness" for the PCR band. Of course, if you clone the 
PCR product ....

Good luck

Peter

Peter Neame
USF and SHCC