Questions on methylation interference assay

Beatriz Gonzalez-Yanes beatriz at pine.circa.ufl.edu
Sat Nov 14 21:01:00 EST 1992


Bionet readers,

While waiting to get a good exposure of my DNAse I footprinting gels 
(3-4 weeks), I decided to try methylation interference.  I basically 
followed the protocol in Current Protocols in Mol. Biol. After 
methylating my end-labelled 250 bp probe, I did as for the gel 
mobility assays, and isolated the unbound and retarded complexes.  
However, recovery was very low and I kenw I was going to have ot 
expose it longer than my DNAse footp. gels.  So I tried doing the 
binding, and then methylating.  Areas of the DNA protected by binding 
proteins will not be methylated (I figured).  For this I used enough nuclear 
extract to shift all of my label.  As  a control, DNA w/o protein was 
also methylated. Then I did as usual, piperidine cleavage, etc.

As a result I got a beatiful ladder, identical for both samples.  A 
friend in the lab says she's seen it done like this before, but has no 
reference.  I thought this way was faster than running a gel 
retardation and exposing overnight, and so on.

Any comments on this will be appreciated. It is not "life or death", 
but I hate trying a protocol that has worked for others, and then 
finding I can't get it to work.

Beatriz.

beatriz at ufpine            beatriz at pine.circa.ufl.edu
Cuando suena el reloj despertador por la man~ana, recuerdo que "al que 
madruga Dios lo ayuda", pero como "nunca es tarde si la dicha es buena"
y "no por mucho madrugar amanece mas temprano", sigo durmiendo.



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