EMSA using ds Oligo nucleotides

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.