Genomic DNA isolation

kang at msvax.mssm.edu kang at msvax.mssm.edu
Thu Aug 24 00:00:42 EST 1995


In article <41ejkc$ger at apopi.u-strasbg.fr>, Francois Nantel <Nantel at titus.u-strasbg.fr> writes:
>In article <saleem.30.00048BF0 at rfhsm.ac.uk> Dr Saleem Mohammed,
>saleem at rfhsm.ac.uk writes:
>>Hi,
>>
>>Seem to be having a few problems with shearing when I am isolating
>genomic DNA.
>>The protocol is the traditional RNAse/proteinase K/phenol extraction
>method.  
>>All the tips for pipettes are cut to ensure minimisation of sgearing
>effects, 
>>but still the problem persists.
>>
>During the phenol extraction, it is important to keep the rotation at
>less than 1 rpm. This was not evident in most of the protocols I read.
>
>Hope this helps
>
>Francois

Hi There;

I exactly do not know the purpose of your genomic DNA purification and from
which organism. However, if the purification is from mouse tail or cell culture
or some mouse organs, and the purpose is doing Southern or PCR, I do not care
too much about the shearing. Everybody knows very well that it is impossible to
purify intact chromosomal DNA. 
The best size you can have by conventional methods may not exceeds 60 Kb. This
is very natural. In my case I am doing vortex after adding phenol or sevag. If
I check my undigested DNA, major band falls around 30-40 Kb, of course with
some shearing. This minor shearing never gave me a trouble for Southern or PCR. 

Rather, sometimes I could not find my DNA after digestion with enzymes. I think
this is the problem of DNase contamination.




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