gel retardation assay
p.r.ashby at dundee.MAPS.ac.uk
Thu Jun 10 11:11:52 EST 1999
In article <375FCF4E.3E37604B at med.kuleuven.ac.be>, Inge Depoortere
<inge.depoortere at med.kuleuven.ac.be> wrote:
> I want to set up a gel retardation assay for NF-kappa-B. Can someone
> give me a good and quick protocol for the preparation of the
> radiolabeled oligonucleotide probe. I will start from a synthetic single
> stranded probe (20bp) and want to have a double stranded labeled probe.
> Can I use the end labeling procedure with T4 polynucleotide kinase? How
> should I make it double stranded. First label and then anneal (under
> which conditions) or first anneal (how to do) with an overhang and then
> label .
first anneal to a complementary oligo: mix equimolar amounts in a small
volume (5-10 ul). Heat to 70C for 10min, cool to RT. End label with T4
PNK. Main reason is that you don't really want to heat hot oligos to 70C
now do you?
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