FLASH gene-mapping a big headache!! HELP!

Heidi Moss hmmoss at MAIL.MED.CORNELL.EDU
Wed Feb 15 16:11:14 EST 1995


Has anyone ever used Stratagene's FLASH non-radioactive gene mapping 
kit?? (It uses labled T7/T3 probes against partial digests to map 
restriction sites). I followed the protocol EXACTLY and I am not getting ANY 
signal (ok, maybe a REALLY faint one after leaving the film in for two days, 
but nothing worth while), not even for the control DNA supplied with the kit!! 
I have performed every test in the book: checked the transfer via re-staining 
gel with EtBr, stripped the membrane and RE-probed for a known sequence in my 
insert with ECL and got a GREAT signal, increased hybridization time, decreased 
washing conditions, increased amount of DNA, played with partial ranges, 
(most of this with the supplied control DNA -- I didn't want to waste my 
sample) and everything to no avail. I contacted the company and they are 
sending me a new kit for my troubles, but I am DESPERATE for any advice. 
Is there a trick that I am missing? My conclusion that it's either the 
probe itself (are oligo probes somewhat tricky?? they come already 
labelled), or something with the hybridization/detection.
ANY ADVICE WOULD BE APPRECIATED!!
Thanks in advance,
-- Heidi




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