Boehringer Mannheim Genius
hamel at ccu.umanitoba.ca
Fri Feb 19 12:27:19 EST 1993
In article <1993Feb16.163644.6884 at cis.uab.edu> RIVERS at IPRSH.DOM.UAB.EDU (Charles Rivers) writes:
>Has anyone tried the Boehringer Mannheim Genius Kit? What has been your
>reaction. We seem to be experiencing a great deal of lane background on
>genomic Southern blots. We use the MSI MagnaCharge and MagnaGraph membranes.
>Has anyone used any other types of membranes? What have been your results?
>Charles and Yan
Have you tried including 0.5% tween20 in your maleic acid wash solution to
be changed 4 times, 15 minutes per wash (to wash off antiDIG antibody).
Also are ALL solutions made up of FRESH distilled/filtered water, filtered
and autoclaved stock solutions? And what about your hybridizations,
stringent conditions for hyb and washing? And long enough pre-
hybridization (an hour in 1% blocking powder/6x SSC/0.5% SDS). Since the
antiDIG is an alk phos conjugate, the substrate will also be
dephosphorylated by ANY phosphatases that may be in the system (we use
CSPD chemiluminescent substrate by Tropix).
This method of detection is more sensitive than P32, especially
since you cannot monitor the background during washes with DIG system
while you can with higher energy beta emitters such as P32 (P33, S35,
etc). Also, I recommend NOT allowing the antiDIG conjugate to bind for too
long to you membrane. The MSI +ve charged membrane works fine for us
(Southerns, northerns and slot blots). There's not any real need to fix
the nucleic acids to the membrane after transfer since membrane is +ve
charged (try it yourself ... ), just air dry (we use 37oC walk in
incubator room, filter sandwiched in Whatman (3mm or whatever) filter
paper, I leave it there until ready to use (at least couple of hours.
Membrane can also be baked at about 120oC, UV treatment (on
transilluminator for 5 minutes) tends to result in increased background,
air drying/baking are better, although I have noticed that UV treatment
allows (slot blot in these cases) ready detection down to 0.1 pg of
plasmid DNA using 380 bp heat denatured DIG labelled (by pcr) insert probe.
If any more info needed just email me a note.
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