losing my plasmid

[Dean Danner Lab]

In article 1806952254130001 at 132.206.172.152, cycs at musica.mcgill.ca (CS) writes:
>Hi!
>
>   I got problem with my plasmid.  I did a ligation of a mammalian
>promoter gene into the luciferase vector PGL3 from promega.  When I picke
>colonies from the plate after transformation and grow them in 5 ml of LB
>to do minipreps everything is fine.  However, when I trying to grow more
>of these bacteria, I'm losing the DNA completely.  I used the XLI-B as an
>host and I'm growing the bacteria with 100 ug/ml of AMP. and 12.5 ug/ml of
>tet.  The bacteria are growing but the DNA is gone.  Could someone tell me
>what's going on and what I can do to solved my problem.
>
>Thank you
>
>CS



Perhaps this is a low copy-number plasmid. I have had similar problems
with pET vectors.  I finally had to breakdown and do a large scale 
PEG prep of the plasmid to have enough to see on a gel, much less do 
do anything with it.

Brett Burkholder
burkhold at gmm.gen.emory.edu