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Procedure
Design complementary oligonucleotides with compatible half sites on the ends (I use BamHI and BglII). Dry down 300 ng of the two purified oligos together and resuspend in 9 m l Q with 1 m l 10X Annealing buffer. Place in a 65℃ water bath for 2 min and cool in 50 ml of the 65℃ water in a beaker on ice. This takes 15-20 minutes.
Mix the following and incubate at room temperature for 30 min:
1 m l of the annealed oligo mixture
5 m l 10X Klenow buffer
25 m l dNTP mix (21 m l Q, 3 m l 10 mM dATP/dTTP dGTP)
5 m l a 32 P dCTP
14 m l Q
1 m l Klenow
Bring up to 200 microliters with TE, phenol/chloroform extract and ethanol precipitate with 0.1 volume of 3 M NaOAc pH 5.2 as well as tRNA. Dry and resuspend in 100 m l TE and count. I usually get 200,000-400,000 cpm per m l.
Generate a table of binding reaction parameters and competitor concentrations. Prepare the gel and running buffer.
EMSA Gel:
8 ml 30% acrylamide (0.8% BIS)
6 ml 50% glycerol
6 ml 10X TBE
40 ml Q
180 m l 10% APS, 180 m l TEMED
Cool to 4℃ along with the appropriate amount of 1X TBE.
Mix the binding reagents in the following order:
10 m l 2X Binding Buffer
3 m l dI/dC
Q to 20 m l (see table)
competitor at 10-20X excess
10-50 fold dilution of the probe
NE usually 2-4 m g is sufficient
Incubate at room temperature for 30 minutes and load directly onto gel with no loading dye. Run the gel at 200V for 4 hours. Note: it is best to pre-run the gel during the 30 min binding reaction.
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