DNA staining in agarose gels(tell me more)

Bernard Murray bernard at elsie.nci.nih.gov
Mon Nov 15 20:00:37 EST 1993


In article <1993Nov15.101702.1 at mcvax4.d48.lilly.com>, dallas at mcvax4.d48.lilly.com writes:
> In article <2c30m2INNdcq at medicine.wustl.edu>, eric at bcserv.WUSTL.EDU (Eric R. Hugo) writes:
> > As a followup to the methylene blue staining discussion
> stuff deleted...
> I discovered that I get 20-50X as many
> positive clones from band isolated DNA when comparing MB
> to ethidium bromide/UV transillumination.
>      Hope that this was of some help
>                               Eric >                               Eric
> 
> Other than the obvious that the UV and ETBr damages the DNA, is there something
> else going on that increases your amount of positive clones??  I'm curious.
> 
> Details on source of agarose...
> Sizes of bands isolated, etc.
> 
> Jim Miller
> Lilly Research labs
> 
> 
> 
> >   Eric R. Hugo, Postdoctoral Research Associate |eric at bcserv.wustl.edu
> >   Dept. of Biochemistry and Molecular Biophysics|finger above for            
> >   Washington University School of Medicine      |public key <-- 
> >   Box 8231, St. Louis, MO 63110_________________| (314) 362-3342

I realise that this drifts from the heart of the topic somewhat but there
are plenty of overlaps...

Concerning the methods used for the purification of DNA in general, and
plasmids in particular -
What's the general feeling about the influence of the method used for DNA
purification upon its suitability for the eventual task?
I purify plasmids for use in mammalian cell transfection studies.  On the
one occasion that we were able to perform a direct comparison we found that
a plasmid purified using a resin (MegaPrep) gave a superior precipitate with
calcium phosphate than the same plasmid purfied by CsCl/ethidium bromide.
Since mentioning this to co-workers I have heard more agreement than
disagreement and have been shown anecdotal reports in the literature that
support this.  In addition to this effect, I am wondering if plasmids
purified from resins/glass are in better shape chemically than those
subjected to ethidium.  Maybe the observations from gel purification of
fragments could shed some light on the subject.
	Any opinions?.....
				Bernard

Bernard Murray, Ph.D. - NCI/NIH
bernard at elsie.nci.nih.gov
bmurray at helix.nih.gov



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