How many times can you re-use a Southern?
nickjacobsen at yahoo.co.uk
Fri Oct 19 09:39:43 EST 2001
i used to do loads of pulsed field electrophoresis southerns and used
to strip, using the same method as you, and rehybridise them around 10
to 15 times. At that point they would start suffering. After blotting
I used to fix the DNA to biorad zetaprobe nylon filters using NaOH
incubation (as detailed in the protocol booklet) followed by baking at
80 degrees C for 2 hours. this seemed to make the blots last longer
(ie more like 15 times). i think its a simple case of the DNA target
becoming slowly washed off by repeated stripping.
hope this helps
John Dixon <jpcd100 at mole.bio.cam.ac.uk> wrote in message news:<191020011029429470%jpcd100 at mole.bio.cam.ac.uk>...
> Hi All, we have a number of replica filters with assorted mouse genomic
> digests, that we use to restriction map each locus of interest.
> Recently our hybridizing bands have started to become very faint, and
> one consideration is that the DNA may be "worn out" in some way, by
> repeated stripping (boiling 0.1% SDS, poured on, cool to RT),
> nucleases, or whatever.
> Can anyone give me a rough idea how many times we should expect to be
> able to re-use these things before they're knackered; once, ~10 times,
> ~100x, indefinitely?
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