Alkaline Transfer... Hybridization too alkaline?

[Josh Neufeld]

In article <3.0.32.19990405150026.00686e6c at pop.mail.nmu.edu>,
  rlara at nmu.edu (Ray Jonathan Lara) wrote:
> Dear all...
>
> I am performing downward alkaline transfer for genomic DNA blot
> hybridizations (and the transfers are great!), however, the hybridization
> signal is just not there.  I was wondering whether or not the alkaline
> conditions (pH ~10-11) after the transfer (even after 2X SSC
> neutralization) inhibits DNA/DNA probe hybridizations?  No matter how much
> 2X SSC I use to neutralize the blot after transfer, the pH is still above
> 10.  By the way, I am using the Boehringer Mannheim DIG kit, if that is of
> any significance to alkaline conditions...
>
> Thanx for any advice in advance.
>
> Jonathan
>

Dear Jonathan, I currently do alkaline transfers using a dot/slot blot
manifold and probe with DIG labeled gene probes.  The DIG bound to the probe
is alkaline labile and so it is very important to wash your membrane
adequately after transfer.  I transfer DNA, rinse briefly in 2xSSC,
cross-link the DNA, then rinse 3 x 5 minutes in 0.5M Tris-HCl, pH 7.0.	I
have not had any problems with this method.  If you have any other questions
regarding prehyb/hyb conditions or probe labeling, feel free to email me.
Josh.

Josh Neufeld (B. Agr. Microbiology)
Graduate Student
Macdonald Campus of McGill University/
Pulp and Paper Research Institute of Canada

Josh Neufeld (B. Agr. microbiology)
Graduate Student
Macdonald Campus of McGill University/
Pulp and Paper Research Institute of Canada

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