lambda dna extraction
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.
Department of Molecular Neuro-Oncology
Rockefeller University, New York
More information about the Methods