Problem with human genomic Southern Blot

Frank O. Fackelmayer Frank at Fackelmayer.de
Fri Oct 4 11:41:33 EST 2002


Hi Eva,
Most probably, you are either not loading enough genomic DNA onto your
gel, or your probe is not hot enough. 

Samples should not be less than 10ug of total genomic DNA per lane (I
usually used 30ug), to have a sufficient amount of target DNA (with
30ug, a unique DNA fragment will not be more than a few picograms).
Using a BAC as a positive control, be sure to dilute it to the amount
you calculate. 
E.g.  a 3000 bp fragment in a diploid human genome (2*3e9bp) is only :

3000/(2*3e9)= 0.0000005 or 0.00005%
when you use 30ug, your amount of target will be only 15pg


As to the probe, I have always tried to get the highest possible
specific activity. My probes (25-100ng DNA) usually labelled to 6-9e6
cpm (Cerenkov counting) by random priming.
This was sufficient to visualize a unique 3kb sequence in 10ug of total
genomic DNA (5pg) after overnight exposure. Be prepared, though, to
expose for several days.

Also, do not hybridize for too short time. Overnight is ok, but 20h is
better unless you use one of the new "express"-type hybridization
buffers (or a homemade high-SDS buffer)

Hope this helps,
Frank



Eva RC wrote:
> 
> Hi to everyone.
> 
> This is my first time joining the bionet.molbio news. I'm working with
> Southern Blot at the moment: my samples are human genomic DNA, and I'm using
> a BAC that includes my critical region as a positive control. My PCR probe
> is around 800 bp long and highly specific, and I label it by random primed
> labelling with 32P-dCTP. I've tried many different hybridization conditions,
> including several temperatures and buffer compositions; different digestion
> times and several restriction enzymes; different ways of treating the gel
> before tranfer; ... and I always get the same result: no band at all for my
> samples and exactly the expected band in the lane with the digested BAC.
> 
> Would any of you have an idea of what might be happening, or have any
> suggestion?
> Than you very much.
> 
> Best wishes.
> EvaRC.



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