lambda dna extraction
mb1gt at silver.shef.ac.uk
Wed Apr 27 03:19:09 EST 1994
Bob Darnell (darnelr at rockvax.rockefeller.edu) wrote:
: 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?
We have been having problems getting cells to lyse in liquid medium.
Although small plate preparations of DNA are possible, our lambda GEM
containing our insert will not lyse E. coli in liquid medium, however long
the culture is left. Different titres of phage have been used, but
without success. Does anyone have any experience of a similar problem,
and a solution? Colleagues have confessed that they were unable to
make phage DNA from liquid lysates, and were forced to use plates,
which are messy and give poor yields of DNA which does not cut well.
Neither Maniatis nor the Promoega protocols manual methods have yielded
lysis. Is it perhaps a problem with our particular clone?
: > --
: > 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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