Alkaline Transfer... Hybridization too alkaline?

Bernard P. Murray, PhD bpmurray*STUFFER* at socrates.ucsf.edu
Mon Apr 5 20:02:27 EST 1999


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



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