mapping the transcriptional start site: HELP!!
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!!!
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