lambda dna extraction

Bob Darnell darnelr at rockvax.rockefeller.edu
Tue Apr 19 13:06:56 EST 1994


In article <2oebn2$noi at mserv1.dl.ac.uk>, "shantaram bharadwaj"
<sb at biotech.ssf.ernet.in> wrote:

> Subject:   Lambda DNA Extraction
> 
> I have been trying to prepare large amounts of lambda fix DNA.The lysate 
> titre is very high.  After removal of cell debris, the phage is ppted with 
> NaCl and PEG,  dissolved in TM buffer and PEG is removed by chloroform 
> extraction.  I then purify phage using an ion exchange column.Phage yields 
> are quite good. I have tried  1. Lysis of phage heads with phenol 2. Lysis 
> with SDS and Proteinase K.The second method gives me better DNA yield than 
> the first. 
> 4.  Since ProteinaseK buffer has high amounts of EDTA, I use sod.acetate
>     pH 7.0 for ethanol precipitation. (Ref.  Maniatis).

> Has anybody else faced this problem?  If so, how did you overcome it?

> -- 
> Shantaram Bharadwaj
> sb at biotech.ssf.ernet.in

I also had great difficulty getting phage DNA to cut several years ago.  I
ultimately traced the problem to excess carryover of a contaminant which I
believe to be EDTA.  Specifically, I was using high concentrations of EDTA
(to help lyse phage coats, which are normally dependent on Mg++ for their
integrity) during proteinase K step; this DNA would not digest at all. 
When I lowered the concentration of EDTA to 2mM, my DNA subsequently
digested well; when I mixed the two preps, I got complete inhibition of
digestion.  This problem might well be circumvented by thorough washing of
ethanol pellets with 70% ethanol; however, I have found no need for the
higher concentration of EDTA and thus don't use it.

Good luck.

-- 
Bob Darnell
Department of Molecular Neuro-Oncology
Rockefeller University, New York



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