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sequencing of miniprep dna

Martin Kennedy mkennedy at chmeds.ac.nz
Tue Jul 19 20:41:56 EST 1994


In article <306tcc$9ah at netnews.upenn.edu>, ljohnson at udcemail.udc.upenn.edu (Lois C. Johnson) writes:
> martin et.al.
> 	Just read you notice on miniprep dna and sequencing.  Thanks!! I
> just had one question.  Do you save the snotty part or not? ARe you adding
> the isoprop &/or NH4Ac & isoprop to the snotty part or the remainder in
> the tube if there is any!!?? 
> 	It may be obvious but of course I didn't get it!!
> 
> Thanks a bunch
> Lois

Hi Lois,

The snotty stuff is pelleted, lysed cells and chromosomal DNA all gunked 
together; i.e. it is garbage - but if you can think of a use for it, maybe we 
can make some money out of it!!!  The plasmids, because they are much smaller 
molecules than the chromosomal DNA, sneak out into the supernatant as the 
cells are lysing, so it is the supernatant you keep.  You can either tip that 
off into another tube into which you've already added isoprop, or just pick 
out the gunk with a toothpick.  I changed from the latter method to the 
former, cos I think it gives slightly cleaner preps, but the latter method 
uses only one tube per prep.  Oh, and don't bother adding any more salt - 
there is enough in the STET, and enough RNA to cause all the nucleic acid to 
crash out.  Don't freeze the isoprop/DNA mix either, as this is likely to 
cause more crud to crash out - just spin immediately.

Have fun!

Martin

NNNN   NN  Martin A Kennedy (E-mail = mkennedy at chmeds.ac.nz)  ZZZZZZZ  
NN NN  NN       Cytogenetic and Molecular Oncology Unit          ZZZ
NN  NN NN           Christchurch School of Medicine            ZZZ
NN   NNNN              Christchurch, New Zealand              ZZZZZZZ
	      Phone (64-3)364-0880  Fax (64-3)364-0750



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