Frank O. Fackelmayer
Frank.Fackelmayer at uni-konstanz.de
Fri Aug 20 09:03:59 EST 1999
soenke behrends wrote:
> Dear netters,
> we are doing genomic southern blots with a
> radioactively labelled 300 bp probe. We see
> good signals with PAC DNA as control and
> a very weak signal with human genomic DNA.
> We could get better results with the Clontech
> Express hyb solution but results are still not
> nice and consistent enough to proceed to large scale
> analysis of many samples.
> From your personal
> experience is the quality of the genomic DNA
> crucial? We have prepared by a standard protocol
> with Phenol extraction (blood leukocytes or
> placenta and heart as starting materials).
> Would a commercially available kit with
> columns help here or is from your experience
> something else more crucial. We use
> 10 ug genomic DNA digested with XmnI which
> already produces a big smear on a standard
> agarose gel. Do you think we should nevertheless
> try to increase the amount of loaded samples?
> We would be very happy about any comment (or
> magic trick) or good working protocol.
> Thanks a lot for your time and help
> Soenke Behrends
The quality of genomic southerns depends on quite a number of
parameters, and any one of them might account for your weak
1. Copy number of the target: If you have a unique sequence, you will
have to apply quite a lot of probe to your gel to see it. In my lab,
between 10 and 300ug of digested total genomic DNA is sufficient to get
a good signal with radioactive probes and overnight exposure.
2. Purity of DNA/complete digestion: DNA must be pure enough to allow a
complete digestion with the restriction enzyme you want to use. In case
of insufficient purity, digestion will be incomplete and the signal will
spread over several (or many) bands.
3. Labelling of the probe: For good sensitivity, probes must be quite
hot. I got best results with either nick-translated DNA (but nick
translation must be quite well titrated with respect to DNase
concentration to really get a good labelling) or random priming (I
prefer this one because it produces consistently good labels). Use no
less than 10 million cpm of probe according to cerenkov counting.
4. Blotting efficiency: blotting efficiency must be good, and can easily
be controlled by staining the gel with EtBr after blotting. No DNA
except for very long fragments (>20kb) must be detectable. Optimizing
blotting conditions is usually VERY effective in getting better signals.
I use a vacuum blotter, and perform acid depurination before the actual
5. Hybridization time and temperature. Time should be longer than for
simple plasmid southerns, at least 12h is good. I have hybridized for up
to 48h and got good signals. Temperature is also significant, and should
be the lowest possible temperature that does not yet produce unspecific
signals. You will have to test that individually for all of your probes,
as AT-rich DNA and CG-rich DNA behave quite different. For AT-rich
fragments, 60 to 65 degress ar a good starting point, while for GC-rich
fragments 68 to 72 degrees are preferred.
Hop this helps,
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