phage prep help really, really needed

Joshua Daniels jbdaniel at facstaff.wisc.edu
Tue Nov 8 18:45:58 EST 1994


Hi-

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,


Josh




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