FLASH gene-mapping a big headache!! HELP!
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,
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