hybridization problems

afc at gnv.ifas.ufl.edu afc at gnv.ifas.ufl.edu
Tue Oct 13 10:36:14 EST 1992


In article <1992Oct12.090837.24582 at gserv1.dl.ac.uk>, ED at micro.uct.ac.za (Ed Rybicki) writes:
>> From:       s882076 at yallara.cs.rmit.OZ.AU (Sean Davidson)
>> Subject:    Re: hybridization problems
>> Date:       10 Oct 92 03:11:10 GMT
> 
>> Further to this discussion about different membranes, I recently began using
>> the Digoxigenin-based system for Northern blotting. Initial attempts used
>> Hybond N+, which is recommended for this procedure, but signal was always
>> virtually undetectable amongst high background. When I began using
>> Zetaprobe (Biorad) instead, results were (are) great.
>>   So do you think this may have just been the batch of Hybond, or why are
>> the results so much different?
> 
> I have seen a lab in the US to whom I had extolled the virtues of DIG,
> fail miserably in many attempts to get Northerns to work in an environment
> where 32P blots had been working well.  They also used to get "negative
> bands" where the RNA should have been.  The local reps were of the
> opinion that they should try the specialist Northern kit with RNAse-free
> everything.  I then came home to find people gaily Northerning with plant
> viral RNA and DNA probes with DIG, with excellent results - and they were
> using Hybond-N, whereas my American colleagues were using AN other brand.
> The answer is, who knows?  Probably one problem is the presence of RNAses
> in the various blocking agents, etc, used with DIG - but then why does our
> Genius kit work with our RNA?  Do we have a different batch of Boehringer
> blocking agent?  Ours is still working, with chemiluminescence, and dye.
> Maybe it'll stop, and we won't know why either...!  Will share protocols
> if anyone is interested.

Our experience was similar to the anonymous US lab.  We compared the
Genius kit to Amersham's chemoluminescence kit, BRL's strepavidin/alk
phos, and P-32.  We always had more background and lower signals with
the Genius kit.  I don't know if it was filters or what... we just
used one of the other that worked.

Andrew Cockburn
USDA

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