EMSA using ds Oligonucleotides
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500 mM NaCl 100 m l 5M NaCl
280 m l Q
store at room temperature
10X Klenow Buffer
500 mM Tris 7.5 500 m l 1M Tris pH 7.5
100 mM MgCl2 100 m l 1M MgCl 2
10 mM DTT 20 m l 0.5 M DTT
0.5 m g/ m l BSA 50 m l 10 mg/ml BSA (NEB)
330 m l Q
store at -80℃ in 50 m l aliq.
2X Binding Buffer (ref: Gutman et al. GENE 110, 1992 pg 197)
20% glycerol 400 m l 50% glycerol
20 mM Tris 7.5 20 m l 1M Tris pH 7.5
100 mM KCl 100 m l 1M KCl
1 mM DTT 2 m l 0.5 M DTT
478 m l Q
make fresh as needed
Poly dI/dC
Make a 1 m g/ m l stock and store in 100 ul aliq. at -80℃
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.
