genomic DNA purification questions

Wiggy A.C.Hilton at bham.ac.uk
Fri Aug 19 03:31:28 EST 1994


In article <Cuqz71.Aor at ncifcrf.gov>, pnh at fcs260c2.ncifcrf.gov (Paul N
Hengen) wrote:

>  In article <9408161553.AA13566 at post.its.mcw.edu>
>  FRED at bmt.mcw.edu ("Frederick Garbrecht") writes:
> 
> > ...When I am purifying genomic DNA from 
> > human lymphoid cells, I have a lot of trouble in the 
> > phenol-chloroform extractions - when I pipette off the upper aqueous 
> > phase, the DNA strands always seem to be attached to the junk at the 
> > interphase...
> 
> This might seem simplistic, but have you tried removing the bottom portion
> first? Take out all the junk, then the good stuff is left behind.
> - Just a thought :-)
> 
> *******************************************************************************
> * Paul N. Hengen, Ph.D.                           /--------------------------/*
> * National Cancer Institute                       |Internet: pnh at ncifcrf.gov |*
> * Laboratory of Mathematical Biology              |   Phone: (301) 846-5581  |*
> * Frederick Cancer Research and Development Center|     FAX: (301) 846-5598  |*
> * Frederick, Maryland 21702-1201 USA              /--------------------------/*
> * /Hey, Cool! -> http://fconvx.ncifcrf.gov:2001/~pnh/info.html <- Hey, Cool! /*
> *******************************************************************************


Have you tried using phase lock tubes? They are quite an expensive way of
forming a barrier between the aqueous and phenol-chloroform phases but you
can do the same thing far cheaper......read on.

Take a syringe and fill it with some silicone vacuum grease and then
autoclave it.

Squirt a small (about the size of a pea) amount of the grease into an
Eppendorf tube and briefly spin down to the bottom of the tube in a
benchtop microfuge.

Add your crude DNA prep to be cleaned up.

Add an equal volume of phenol-chloroform (NOTE: IT MUST BE
PHENOL-CHLOROFORM AND NOT SATURATED  PHENOL)

Mix, and then spin again for about 2 mins at 13000rpm to separate phases.

This method is so good that you can simply decant the aqueous phase off the
top of the silicone grease interface with not a hint of crud.

E mail me if you need further instruction/advice on this technique.

Good luck

Wig.
-- 
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      ** It's because I come from Wigan that my name is Wiggy,**
      ** and nothing to do with my head................Honest!**
      **                                                      **
      **                 A.C.Hilton at bham.ac.uk                **
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