hybridization problems

Ed Rybicki ED at micro.uct.ac.za
Mon Oct 12 05:26:02 EST 1992


> 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.

 ____________________________________________________________________
| Ed Rybicki, PhD             |    "Now you've got the hang of it    |
| (ed at micro.uct.ac.za)        | There's nothing you can't do with it |
| Dept Microbiology           |        If you're very into it        |
| University of Cape Town     |         You can't go wrong...."      |
| Private Bag, Rondebosch     |                                      |
| 7700, South Africa          |               -Mad John              |
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