mapping the transcriptional start site: HELP!!

Heidi Moss hmmoss at MAIL.MED.CORNELL.EDU
Sat Jul 29 11:37:05 EST 1995


Greetings to all of you helpful netters!!
I am using Ambion's RPA kit to map the transcriptional start site of my 
gene. Here is my question: I am using a 300bp probe (prepared by Ambion's 
In vitro transcrption kit) that spans 200bp of the 5'utr of my cdna clone 
(farthest 5'end determined via RACE) PLUS an additional 100bp of possible 
basal promoter (gc-rich, numerous sp1, NFE1 sites, etc -- BUT CAAT-less 
and TATA-less, which is not unlikely for this gene). I am wondering what 
would be the BEST acrylamide gel conditions to determine the 
transcriptional start site TO THE NUCLEOTIDE. I am suspecting multiple 
sites, typical of TATA-less, gc-rich promoters, so I want to be as 
accurate as possible.
?? gel %, gel length, gel width, gel thickness, etc.?????
Do you think this probe is adequate?
Any imput for this experiment would be GREATLY appreciated!!!
			Thanks!
				-- Heidi




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