genomic southerns

Robert M. Mihalek mihalek at FORMULA1.smtp.anes.upmc.edu
Fri Aug 20 09:26:32 EST 1999


In article <7pjfq9$hdu$1 at rzsun02.rrz.uni-hamburg.de>
behrends at plexus.uke.uni-hamburg.de (soenke behrends) writes:

> 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 process thousands of genomic DNA samples from very crude lysis of
mouse tail biopsies. The DNA is very dirty (usually brown or even black
color) and we never do phenol extractions. So, I think dirty DNA is
okay.

The enzymes we use are usually the most vigorous enzymes available,
such as BamHI, EcoRI, BglII. We'll use around 10 ug of DNA and let the
digests go for a couple hours at 37C. We have had problems with partial
digests with Eco RV.

If you see a smear all the way down the lane and also see the faint
bands representing repetitive DNA fragments, then you know that the DNA
is digesting well.

One suggestion would be to see what kind of signal you can get with
another enzyme.
You could also try to reduce hybridization temp to see if you can more
probe to stick to the target, or reduce washing temps/ increase salt.
Try a 3X SSC wash at 65C. If there's too much background, drop down to
1.5X SSC.

Good Luck!


Bob Mihalek=================================================
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