EMSA optimization?

z z-suldan at ski.mskcc.no.spam.org
Mon Jan 12 09:08:40 EST 1998


> On 9 Jan 1998, Mirkka Herranen wrote:
> 
> > hi, 
> > does anyone have an idea what i should do to overcome this problem
> > in EMSA (gel retardation):
> > after end-labelling the DNA-fragments i heat the fragments to 100oC and
> > let them cool slowly to make them 2-stranded again - but somehow
> > they seem to stay at least partly in single-stranded form !
> > could it be that there's something wrong in the buffer? would the
> > purification of the probe DNA (by running a gel and cutting the
> > 2-stranded DNA) help? also, if someone has a good method to do EMSA
> > i would really appreciate to have the protocol.
> > 
> > thanks a lot!
> > 
> > mirkka
> > 
> > mirher at utu.fi
> > Univ. Turku, Plant Physiology &Mol Biol, Finland
> > 
> > 

I have my doubts that there is enough salt in the PNK buffer to get great
annealing. Why not anneal a high concentration of oligo in high salt (I'll
get you the exact recipe we use if you want), dilute the oligo and THEN
end-label. Should be MUCH cleaner that way.

Alternatively, you might aneeal and then klenow fill in or design a
hairpin oligo. These should be even cleaner.

Good luck,
Zal



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