genomic southerns

Morris F. Manolson mm6y+ at andrew.cmu.edu
Fri Aug 6 12:39:49 EST 1993


>David Stillman wrote a long time ago :-)..
>>We have been having problems with restiction digests for southerns, and are
>>hoping some one will be able to help us.
>.....
> 
>> The problem is that our DNA
>>preparations (made by the method of Hoffman and Winston Gene
57:267-272 >(1987))
>>will not cut.  This appears to be a strain specific effect, since DNA preps
>>from other strain backgounds cut fine.

My opinion is that 1) ANY genomic DNA can be cut no matter what
type of method you use, and 2) the problem is always protein
bound to the DNA and 3) the solution to DNA that will not cut
is to

1) resuspend "BAD" DNA in 300 ul TE pH 8.0
2) add 8 ul of 2 mg/ml RNAase- 1 hour at 37 C
3) add 6 ul 20 mg/ml proteinase K, 30 min at 65 C
4) repeat step #3 one more time (what the hell eh?)
5) Extract FOUR times with phenol:chloroform:IAA or untill
you see NOTHING at the interface
6) precipitate DNA with salt and EtOH

After that the DNA will always always cut.  Sorry for taking so long
to post this David, hope it still helps.  Morrie (if it don't cut, protease 
the hell out of it) Manolson
  
******************************************************************************
Morris F. Manolson                     Tel:  416-813-6662  (office)
Division of Cell Biology                     416-813-5594  (lab)
Hospital for Sick Children                   416-813-5028  (FAX)
88 Elm St., McMaster building        email:  Morrie at resunix.ri.sickkids.on.ca
Toronto, Ontario, Canada
M5G 1X8



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