E. coli restriction systems

Duncan Clark Duncan at genesys.demon.co.uk
Thu Sep 29 08:36:39 EST 1994


In article: <9409272134.AA07693 at lenti.med.umn.edu>  keithr at LENTI.MED.UMN.EDU ("Henry Keith Reding ", 
MicroBio) writes:
> 


> 3.  Are other E. coli strains available that are further deleted in 
> restrictions systems?
> 

What you want is an E.coli strain that is mcrA- mcrb- mcrf- mrr- hsdr-. Such 
strains are as restriction minus for all known systems so far. A number 
are available from NEB ER???? strains (see back of NEB catalogue), 
Stratagene - XL1BlueMRF, Invitrogen - TOP10f', GibcoBRL - DH5mcr. 
have you looked in your Clostridium for an RM system. It is actually 
quite simple. Grow up a 100 mls, spin down and resuspend in 
10ml 20mM KPi (pH7.4), 5mM mercaptoethanol, 10% glycerol. Sonicate, 
microfuge a portion and assay 10, 3 and 1ul of 
supernatant with 3ug lambda DNA (methylated and unmethylated) in MED RE
buffer in say 50ul. Incubate at 37C for 20 mins and run on a gel. Repeat
with other viral DNA's ie Adenovirus2, T7 etc. If you get too much smearing
then reduce amount of supernatant assayed etc Any cutting into 
discrete bands indicates a restriction enzyme. Clostridia have been 
shown to have various RE systems including iso's of Sau3A I, Fnu4H 
I, Asu II, Hha I, FnuD II, Pst I, Bcl I and EcoR II.

Duncan   
-----------------------------------------------------------------------------
Duncan Clark                        | Internet:    duncan at genesys.demon.co.uk
G4ELJ                               | Compuserve:  100015.1406 at compuserve.com
-----------------------------------------------------------------------------




More information about the Microbio mailing list