Request for Help: single stranded phagemid DNA preps.

[Al Binnie]

(David Steffen) writes:
>
>  We are attempting to prepare single stranded phagemid DNA and having
>problems...
>
>...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 have had very similar problems in the past.  Even though our phagemid is 
extremely stable in cells (it's next to impossible to cure cells of the 
plasmid by growing without ampicillin), we find that if you subculture the 
phagemid-containing cells too many times the ratio of helper:phagemid goes way
up.  This happens fairly quickly.  By the time we sequentially grow 5-10 liquid 
cultures to saturation there is practically no production of phagemid.  The
other phenotypes conferred by the phagemid are not affected.  The problem 
was solved in our case by isolating phagemid from the cells which no longer
produced ss-phagemid and retransforming the host strain.  The newly transformed
strain invariably had a very low helper:phagemid ratio.  Thus it seems that
some kind of imprinting (possibly a chromosomal mutation?) takes place in 
sequentially cultured cells which blocks efficient production of ss-phagemid.
I don't understand the phenomena, but you can get around it by avoiding 
sequential culturing.  We now save the original colonies picked after 
transformation in liquid media and dilute cells out from that stock over and 
over again.  The regeants in our case are;   cells:     TG1
					     phagemids:	Bluescript derivatives
					     helper:    M13K07


Hope this helps.
                       al binnie......
                                      Purdue University
                                      Department of Biochemistry
                                      Internet: binnie at aclcb.purdue.edu
                                      317-494-1658 (W)