Request for Help: single stranded phagemid DNA preps.

David Steffen steffen at mbcr.bcm.tmc.edu
Mon Oct 19 16:28:37 EST 1992


Hi, y'all!

  We are attempting to prepare single stranded phagemid DNA and having
problems.

  The idea is that a bacterial stock containing a plasmid containing
an m13 origin of replication is infected with a defective m13 helper
phage.  The defective helper is inefficient at packaging its DNA.  The
helper phage not only replicates its own DNA, but induces single
stranded replication of the plasmid via its m13 origin of replication.
Because of the packaging defect in the helper, the plasmid DNA gets
packaged, released into the medium, and viola!  Or so goes the theory.

  This used to work for us.  More recently, all we recover is the
helper.  We have checked both genetically (amp selection) and
biochemically (plasmid prep) and the bacteria still have the plasmid.
The phage stock has not been propagated, so we don't see how it could
have "back-mutated".  We have not knowingly changed any conditions.
Thus, we are at a loss.

  We are using a Biorad kit, including the bacterial host CJ236, and
the defective helper 13K07.  Our kit is about 4 months old.

  Any thoughts?

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



More information about the Methods mailing list