phage prep help really, really needed

[Shahram Mori]

Joshua Daniels (jbdaniel at facstaff.wisc.edu) wrote:
: 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:


: 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.


It's possible that your DNA is still bound to the white goop. Why don't you
try at this point ( prior to phenol chloroform stage) to incubate the
white stuff with TE at 37C for 10-15 minutes. This might entice the DNA to
leave the goop.


  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.  


: Josh


It would be a good idea to also do a dot blot after each stage to keep
track of your DNA. This might tell you which stage is the culprit for the
loss of DNA ( if in fact there is one).
Cheers

 --
Shahram Mori					   _/\_
Program in Molecular Biology			  _\  /_
Dept. of chemistry and Biochemistry Box 3C	  \_  _/
NMSU  Las Cruces NM				    ||
88003