LB vs. TB?

T. J. Murphy medtjm at bimcore.emory.edu
Tue Nov 16 09:22:05 EST 1993


In article 9371 at alw.nih.gov, bernard at elsie.nci.nih.gov (Bernard Murray) writes:
> 
> Hello,
> 	Earlier in this thread it was implied that cultures grown in TB are
> unsuitable for mini-preps by alkaline lysis.  So, how do you prepare your
> litre preps from this medium?  Can you use methods based upon glass/resin or
> is CsCl essential?
> 	(Anything to get more plasmid for less effort)
> 				Bernard
In reply...
We use neither glass milk nor CsCl.  After alkaline lysis of large scale cultures
(Sambrook et al., 2nd edition, p 1.38) we purify the plasmid DNA by LiCl/PEG precipitation
(ibid p. 1.40).  We don't do this for minipreps,  maybe that's why the quality differs.

We are making our living by transfections of mammalian cells, and the DNA prepared
in this way is fine for transfections, plus the yield is quite high, so fewer preps
per transfection series.  Yield suffers in our hands if we do CsCl banding, plus all
the EtBr and dialysis is too much toxicity and work for little additional advantage.

We do reporter assays.  We do get some prep-to-prep variability by this method which
can be frustrating, but we also see the same variability with CsCl-banded DNA.

I've never tried the glass milk preps so have no comments on this method other than
I am sure our shelf reagents are cheaper per prep than the kits available.



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