IN-VIVO foot-printing

Angelo Gunasekera angelo at phoenix.Princeton.EDU
Thu Oct 22 07:43:56 EST 1992


I have been using invivo foot-printing protocol to identify protein
binding sites in the E.coli chromosome.  I have been getting really good
footprints and can identify the regions in the E.coli chromosome.  The
main problem I am consistently getting is that the DMS (dimethyl
sulfate) treatment of the E.coli cells also seem to produce methylation
or strand break at C's and T's in addition to expected sites at A's and
G's (by the way I use a 32p labelled primer to do the PCR).

I am wondering whether anyone in the net has similar problem in doing
the in-vivo foot-print in the e.coli chromosome or can suggest me their
experties to avoid this non specific strand scission.

I have already tried the following without any sucess

1. Different concentration of DMS and the duration of the DMS treatment
2. Avoided the pipiridine treatment and expected high temps. in the PCR
   cycles to break the strands at modified bases
3. Different OD 600 of E.coli cells before the DMS treatment.
4. Tried many different protocols to purify the methylated E.coli
   chromosome.

I do not have any problem when I try to footprint the plasmids
containing the E.coli chromosomal copy 

Thanks in advance for your replies.
 
Angelo Gunasekera,
 Dept. of Chemistry, Princeton University
Angelo at bigvax.princeton.edu Angelo at bigsgi.princeton.edu
                eton.edu, Angelo at bigsgi.princeton.edu



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