promoter-walking

gordon allison goa at aber.ac.uk
Tue Sep 17 10:29:50 EST 1996


Any advise would be welcome, but please use my email or else I might miss 
it. I am screening a couple of sheep genomic libraries for the promoter of 
a sugar transporter. I am getting clones but it is becomming apparent that 
there is a lot of intron. I recently became aware of Clontechs promoter 
finder kit which walks in from the extreme 5' end of the cDNA sequence by 
PCR. It sounds like everything else"a piece of cake", but I bet it is'nt.

If I want to do this I wil probably have to make my own genomic libraries 
for amplification. Clontech offer 5 in their kit. The idea is that 
restricted genomic fragments are ligated to an adapter oligo which is used 
as a primer binding site in PCR in conjunction with a gene spec. primer. 
Just as in RACE the 1st generation products are something of a smear but 
this is resolved by a 2nd round of PCR using nested primers. Clontech 
advise that a long-range poymerase mix is used (Vent+rTth)with a stringent 
start. I have asked them for advise but I think that it sounds so nifty 
that I really must give it a go.

I know that someone out there will be saying"use the human kit available 
and do primer analysis with that", but I want the sheep promoter OK!

Please write back if you have any pearls of wisdom to share. Thanks in 
advance. Gordon.




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