IUBio

How to circumvent DNA restriction in a bacterial host?

Amy Amy.Verhelst at mailgw.surg.med.umich.edu
Tue Nov 21 17:00:15 EST 1995


Heat shock subdues the type I restriction system in coli in a way that
lets you get some DNA in.  You could also try methylating DNA in vitro
with some available methylases (this would probably only work if type II
endonuclease is responsible, and might call-in problems with dcm-type
restriction)  Good luck!

In article <48s9u1$rvt at nntp.ucs.ubc.ca>, bina at unixg.ubc.ca (Jim) wrote:

> Hi,
> 
> I am trying to do gene replacement in a gm(-) bacteria that contains an 
> uncharacterized restriction/modification system(s), however, I am not able to 
> get recombinant transformants via electroporation or CaCL2 methods.  I assume 
> the problem is DNA restriction since I had a tough time cloning DNA from this 
> organism.  I tried using several different R/M E. coli strains for
preperation 
> of DNA without success.  Does anyone have any suggestions or ideas on
how I can 
> get arround this problem?
> 
> Thanks Jim.



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