phage prep help really, really needed
jbdaniel at facstaff.wisc.edu
Tue Nov 8 18:45:58 EST 1994
I've been trying to do plate lysates for DNA extraction for some embl 3 clones
that I must start mapping!! I've been stuck at this part of the process for a
month now and I'm going nutso.
I've been using:
Top agarose with tryptone, NaCl (lambda medium, per current protocols)
Lambda bottom agar.
If I get nice totally lysed lawns after o/n incubation I put 13 ml SM (for a
150 mm plate) for 5 hrs and use that as the lystate.
For getting the DNA I've tryed the following:
ppt with PEG 8000, NaCl after a pre-clear of the lysate with CHCl3 and DNAase
incubation to get rid of Host DNA (LE392 cells for host)
After the PEG ppt, I then clear the PEG with CHCl3 extractions and then crack
the phage with phenol and then do more CHCl3 extractions
Alternatively, instead of the PEG ppt I simply pelleted phage in an
ultracentrifuge at 132,000 X g for 1.5 hrs. I got a nice translucent pellet
which I resuspended in .05 M pH 7.5 Tris. Then I added phenol to crack the
phage. Upon doing this, I got a white ppt which I assume was phage capsid
protein. The aq phase was phenol ext, and CHCl3 ext several more times and
then I tried to ppt with 3M NaOAC and 2.5 volumes EtOH (the usual). No dice.
I got a pellet, but sure wasn't phage DNA.
If you can offer any advice so far as what has worked for you (short of a CsCl
gradient) please let me know.
sincerest thank yous,
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