plasmid curing

[qwqwe2000]

Hi,I want to cure plasmids from Gram positive bacteria.Does anyone know some methods that is nontoxic and efficient?
Thank you.

[gandalfthegrey]

When you mean "cured" you mean removal of the plasmid?
The definition I know is: curing is the elimination
of a plasmid from its host cell.

If that is so, I found this protocol which was from an
old bacteriologist and it worked for a friend of mine
(who did it once though):

1. Grow bacteria with plasmid overnight. use approximately 2x10^9 cells/ml.
2. Dilute them to 1x10^4 cells/ml in LB, 0.1M Tris, pH 7.6, 20ug/ml acridine orange.
3. Grow overnite in dark.
4. Replica plate to find "cured" bacteria.

Also, you can use Novobiocin as it interferes with gyrase
(as does Nalidixic acid and several other agents -
Oxolinic Acid) which is needed for plasmid replication.

ethidium bromide has been used along with other intercalating
dyes.

see this paper for a new method:
SpringerLink - Journal Article

It also helps to culture the bacteria at higher temperatures such
as 42C or 45C.
See this paper for more information on temperatures:
Interspecific Transfer of Streptomyces Giant Linear Plasmids in Sterile Amended Soil Microcosms

Good luck

[curious student@biotech]

hi, can anyone help me about plasmid curing of gram positive bacteria using ethidium bromide?