From: steffen@mbcr.bcm.tmc.edu (David Steffen)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Summary of phagemid prep suggestions - THANKS!
Date: 6 Nov 92 23:48:24 GMT
Organization: Baylor College of Medicine, Houston, Tx
NNTP-Posting-Host: mbcr.bcm.tmc.edu


  A week or so ago, we posted a request for help solving a problem we 
are having making single stranded DNA from a phagemid.  This is a brief 
summary of the (many) suggestion we received.  The edited suggestion 
themselves were 6 pages (14k).  These contain more information than I 
could include here; if anyone wants, I can email these to you, or even 
post them if the will of the net so dictates.

BASIC METHOD:
------------

A bacterial stock containing a plasmid containing an m13 with an M13 
origin of replication (a phagemid) is infected with an m13 helper phage 
defective helper in packaging its DNA.  The helper phage not only 
replicates its own DNA, but induces single stranded replication of the 
phagemid. The phagemid DNA gets packaged and released into the medium.

Because M13 is a male-specific phage, the bacteria also needs to contain 
an F factor.  Finally, because we are using the single stranded DNA for 
site directed mutagensis, we need to use a host strain of bacteria 
(CJ236) which incorparates deoxyuridine into DNA in place of thymidine.

There are a number of ways this system can be used to generate phagemid 
particles.  The way we use it is to stora a stock of bacteria contain 
the phagemid and when we need DNA, infect it with the defective helper 
overnight and harvest the pahge the next moring.

OUR PROBLEM:
-----------

After several successful preps, we now find that we produce large 
amounts of helper DNA, but no phagemid DNA that we can see on a gel.  We 
can detect the phagemid in the bacterial stock both by AMP selection and 
by plasmid minipreps.

THE SUGGESTIONS:
---------------

(# in [#] at the end of line = # of people suggesting this)

Change the host strain of bacteria; specifically avoid CJ236 [3]
  (We cannot do this in our application, but the general rule is do not
   use CJ236 unless you have to.)

Check for maintenance of F' by selecting with chloramphenicol [3]
  (We do not think this is our problem as we still make helper.)

Check for loss of helper phage (select with KAN)
Use VCSM13 helper phage.
Make sure helper phage is not wild type
Plaque purify M13K07 helper phage stock

Grow bacteria in buffered, rich broth.
Grow pahge for 16 to 18 hours after infection.
Leave out kanamycin.
Vary titer of helper.
Results depend on the sequence of the phagemid insert; Try reversing the
   orientation of the insert.

Check for loss of phagemid (select with AMP)
Low yield of phagemid suggest a flaw in your technique, no phagemid
   suggests a genetic problem.  To measure a low yield of phagemid,
   titer the phagemid by selecting infected bacteria for AMP resistance.

Make a fresh transformant [4].
  (Nobody seemed to know why this makes a difference, but apparently it
   does.  Unfortunately, we tried this and it didn't solve our problem,
   but it may solve yours.)

THE RESPONDANTS: *THANKS*!!
---------------

Michael Benedik                              INTERNET: Benedik@uh.edu
Dept. of Biochemical & Biophysical Sciences  PHONE:    743-8377
University of Houston                        BITNET:   Benedik@uhou

jgraham@bronze.ucs.indiana.edu Tue Oct 20 01:04:20 1992

MBJSC@uk.ac.daresbury.dlvh (Chris Clegg)
PHLS Centre for Applied Microbiology and Research,
Porton Down, Salisbury U.K.

JRJ@vaxtm1.rtpnc.epa.gov

Phil Watson
Dept of Medicine, Northern General Hospital, Sheffield UK
bt2pw@uk.ac.sheffield.primea - mbpfw@uk.ac.daresbury.dlvh

preissj@clvax1.cl.msu.edu (Brian Smith-White)

Al Binnie
Purdue University Department of Biochemistry
Internet: binnie@aclcb.purdue.edu    TELEPHONE: 317-494-1658 (W)

jgraham@bronze.ucs.indiana.edu (Jim Graham)
Biology and Chemistry Depts., Indiana University -Bloomington

BHANDARY@wccf.mit.edu
-- 
David Steffen
Department of Cell Biology, Baylor College of Medicine, Houston TX 77030
Telephone = (713) 798-6655, FAX = (713) 790-0545
Internet = steffen@bcm.tmc.edu