Problems with Southern Blotting

Dr. Hiranya S. Roychowdhury hroychow at nmsu.edu
Mon May 17 18:48:02 EST 2004


Alicia,
While some targets will light up in as little as 10ug digested DNA, others may 
indeed need 80ug or more.  The first thing that one needs to look into while 
thinking of doing a genomic Southern is the target to total DNA ratio; in 
other words, you need to have some idea about the C value, the length of your 
target, multiplicity (ploidy level) of the target sequence (if any), etc. So, 
before doing a Southern transfer of genomic digests, you may want to do a 
simple dot blot analysis with increasing (or decreasing) amounts of your 
genomic DNA to determine the approximate minimum amount that you should be 
able to load on the gel after digestion.  Also, remember that the 
spectrophotometric estimation of genomic DNA is not accurate due to its size. 
So, you should be estimating the digested DNA instead for greater accuracy.  
Finally, load about 130% to 200% of the minimum required as determined from a 
dot blot.

Rest needs to be worked out in your hands at your bench!

Hiranya



Quoting Alicia Subtil <aliciasubtil at hotmail.com>:

> Hi everybody,
> 
> I send this message because I am know setting up the coditions to
> perform Southern Blotting. Well, the main problem is that I can't
> obtain any result with my digested genomic DNA. I have tried the probe
> with positive and negative controls that are digested plasmids, but I
> don't have any signal with the genomic DNA. I tried it with 15 ug of
> genomic DNA but someone told me that he uses 80 ug of DNA!!! This is a
> lot of DNA to be ran in a normal agarose gel. What is the amount of
> DNA that is necessary to obtain a good signal of a single copy gene in
> a Southern Blotting?
> 
> Thanks in advance:
> 
> Alicia Subtil
> Lab. Chromatin and Gene Regulation
> Centre de Regulaci󠇥n򭩣a
> Barcelona (Spain)
> 


-- 
Hiranya S. Roychowdhury, Ph.D.
Coll. Asst. Professor,
Molecular Biology,
Dept. of Chemistry & Biochemistry
Rm# 336, Chemistry Bldg.; MSC 3MLS
New Mexico State University
Las Cruces, NM 88003
---



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