Metylation-free strain ?

Stephen Amato amatos at hermes.bc.edu
Sun May 21 10:25:59 EST 1995


In article <3pfrij$rlj at columba.udac.uu.se>, seab at nucleus (Stefan
Baeckstroem) wrote:

> How do I know if a strain metylates my plasmid or not ?
> I'm using JM109 and DH5alpha.  
> --
>                 Stefan Backstrom
>                 seab at nucleus.ibg.uu.se
>                 http://www.ibg.uu.se/~seab


                                  
JM109 and DH5alpha strains are dam plus strains which means that certain
bases are methylated in the DNA.  To demonstrate that you plasmids are
indeed methylated you could separately digest 100-200ng of your plasmid
with the restriction enzymes MboI and DpnI and run the reactions out on an
agarose gel along with an undigested control.  These enzymes have a 4 base
recognition site(GATC).  MboI will not cut the sequence if it is
methylated whereas DpnI will cut the sequence.

amatos at bc.hermes.edu



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