curing plasmids

Takashi Hashimoto hasimoto at bs.aist-nara.ac.jp
Tue Nov 15 23:43:55 EST 1994


I obtained the following message for my question.

---------- Forwarded message ----------
Date: Tue, 15 Nov 1994 17:52:30 -0500
From: rogers elizabeth <erogers at umbc.edu>
To: bionet.molbio.methds-reagnts at umbc.edu
Subject: re curing plasmids

Try growing cells in nutrient broth of choice overnight with 10 
micromolar ethidium bromide or 30-75 micrograms/ml acridine orange.
These amounts were used to cure high copy plasmids in Bacillus species 
and you will have to get levels that won't kill your cells.  Wash cells 
before plating on agar, then screen.  The dyes are thought to bind to the 
supercoiled DNA and interfere with replication of the plasmids. The 
numbers above are from a very old notebook from my predecessor, so I 
can't vouch for the numbers myself.  I have also heard that very low 
levels of SDS can be used which is thought to interfere with the 
replication by preventing binding to the membrane.  This may be safer 
than the dyes which can be mutagenic.  Hope it helps. 
***********************************
Takashi Hashimoto
Graduate School of Biological Sciences
Nara Institute of Science and Technology
8916-5 Takayama-cho, Ikoma
Nara 630-01, Japan
e-mail: hasimoto at bs.aist-nara.ac.jp
fax: (07437)2-5489
tel: (07437)2-5481
***********************************




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