gel retardation assay

Peter Ashby 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:

> Hi,
> 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?

Peter

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Peter Ashby
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