methylation and promoter analysis

Saroj Mathupala mathu at WELCHLINK.WELCH.JHU.EDU
Sat May 21 18:58:13 EST 1994


Hi Bio-Netters, we are using E. coli JM109 for isolation of
promoter-reporter plasmid constructs for transfecting mammalian cells for
transient reporter gene expression studies. Our question is this-since
most E. coli strains that are used to isolate plasmids for such studies
have methylase activity (dam, dcm, and/or EcoK methylase activities), does
that affect/intefere with the promoter analysis studies once the plasmids
are transfected into a mammalian cell line? If so, how long should one
wait before starting the reporter assays (that is, how long does it take
for the 'demethylase/s' in mammalian cells to fully deprotect the
methylated DNA)? Literature currently available indicate that for
completely methylated DNA, it takes between 48 to 120 hours before the DNA
is fully deprotected. Any suggestions or helpful hints would be 
gratefullu appreciated. Thanks in advance, Mathu.




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