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Solutions

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.