Dear Microbiologists,
I am a molecular biologist who specializes in genetics and
recombination of mammalian cells. I have a peculiar
problem I am sure one of you must have run across before
and conquered.... I have a plasmid that rearranges when
it is grown bacteria in large volume cultures!
Specifically it is a plasmid based vector for gene
transfer that is 22,000 bps... yes 22kb!
We have noticed in our lab that it is difficult to grow
up large culture volumes of E.coli(DH10b) containing this
plasmid without rearrangement. Usually I pick a pure
colony and then streak it(on ampicilin plates),two more times
each time picking an isolated colony and preping the
DNA to make sure it is the right size (and the right
RE sites). Then I pick the colony and plate it on a replica plate
and then start a 5 ml LB broth(+amp) of the colony
( so I have a replica of the colony I am growing).
Then I prep the DNA and do the appropriate RE digestions
the next morning.
At the same time that morning...
I have started a 5ml LB(+amp) culture of the same colony from a
pick from the "replica" plate. If the colony has the
right plasmid I start 4X250ml LB(+amp) cultures overnight
using the 5ml LB culture that I started that morning
such that I have kept the bacteria in log phase.
The problem is when I prep the DNA (alkaline lysis+ phenol
chlorophorm) I get either multiple plasmid as well as my
plasmid or I get a truncted plasmid that has lost 5-10 Kbp.
The 5 ml cultures do not exhibit this. Even if I use one
5 ml culture to start another I get the right molecular
weight plasmid and no rearrangements?!?!
I really don't know what to do other than prep several
hundred mini preps from 5 ml cultures! I need 1mg of DNA
so this could take a month!! I have tried DH5a which are
rec- and I get the same thing!!!
Hopefully someone knows a trick, or has had this
problem before...
Thank you for responding in advance
Graham Dellaire
Mcgill Univeristy
Exp Med.
Research
Red Cross
Montreal, Quebec, Canada
fax (514) 525 0881
voice (514) 527 1501 ext. 175
E-mail popa0206 at po-box.mcgill.ca
or B2xe at musicb.mcgill.ca
