in situ hy help

colossus... s535290 at aix1.uottawa.ca
Tue Aug 26 12:36:25 EST 1997


Hi all,

I recently shipped some plasmid DNA to a facility that then takes the DNA
and performs in situ hybridizations to Drosophila polytene chromosomes.
The linearized DNA is used "to give a random-primed, digoxygenin-labelled 
probe.  We visualize the hybridized material with Boehringer Mannheim's
colour detection system". I heard back from them and they mentioned that
they were not getting any labelling of my DNA. Their controls were working
great, and I have no reason to doubt them. My DNA is Quiagen prepped
and isolated from JM105. Since I knew the Dig method might be fastidious,
I took great care to wash the column a few more times than mentioned in
the Quiagen protocol. The DNA sequenced beautifully on an automated
sequencer (ABI 373 using the FS dye terminators), and it also cuts well
with restriction enzymes. They mentioned that they prepare their DNA using
the Promega kits. If you are doing in situs using a similar system, how do
you prepare your DNA ?  I just want to know if switching to Promega will
alleviate the problem, or if it is a different problem altogether,

thanks in advance,

Ed




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