Help! how to get rid of plasmid?
kawalekm at earthlink.net
Wed Apr 11 10:55:02 EST 2001
>I've been doind ET cloning in a BAC host strain. To express RecE/T, we used
>pBADabg (tetracyclin version) from Francis Stewart. We got the recombination
>we wanted. Now, we need to recover the recombined BAC DNA.
>The problem is that there's too much pBADabg and I can't get rid of it.
>I've restreaked the E. coli without tetracyclin a few times. pBADabg
>persists. Does anyone know how to cure this plasmid?
The process you are refering to is termed "curing a plasmid". Some of the
earlier molecular biology literature from the late 70's or early 80's covered
curing. I myself have "cured" plasmids from E. coli, Agrobacterium, and
Psuedomonas. This usually entails culturing cells at an elevated temperature or
under low phosphate stress for an extended period of time; a week or two. For
E.coli, try incubating it at 42-45oC for that period of time. There are other
more envolved protocols involving the use of ETBR or other Acridine Orange. Go
to the literature and find something relevent to the strain you are working
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