EMSA using ds Oligonucleotides
10X Annealing Buffer
200 mM Tris 8.0 200 ml 1M Tris pH 8.0
10 mM EDTA 8.0 20 ml .5M EDTA pH 8.0
500 mM NaCl 100 ml 5M NaCl
280 ml Q
store at room temperature
10X Klenow Buffer
500 mM Tris 7.5 500ml 1M Tris pH 7.5
100 mM MgCl2 100 ml 1M MgCl2
10 mM DTT 20ml 0.5 M DTT
0.5mg/ml BSA 50 ml 10 mg/ml BSA (NEB)
store at -80° C in 50 ml aliq.
2X Binding Buffer (ref: Gutman et al. GENE 110, 1992 pg 197)
20% glycerol 400ml 50% glycerol
20 mM Tris 7.5 20ml 1M Tris pH 7.5
100 mM KCl 100ml 1M KCl
1 mM DTT 2ml 0.5 M DTT
make fresh as needed
Make a 1mg/ml stock and store in 100 ul aliq. at -80° C
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 9ml Q with 1 ml 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:
1ml of the annealed oligo mixture
5ml 10X Klenow buffer
25ml dNTP mix (21 ml Q, 3 ml 10 mM dATP/dTTP
5ml a32P dCTP
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 100ml TE and count. I usually get 200,000-400,000 cpm per ml.
Generate a table of binding reaction parameters and competitor concentrations. Prepare the gel and running buffer.
8 ml 30% acrylamide (0.8% BIS)
6 ml 50% glycerol
6 ml 10X TBE
40 ml Q
180ml 10% APS, 180 ml TEMED
Cool to 4° C along with the appropriate amount of 1X TBE.
Mix the binding reagents in the following order:
i) 10ml 2X Binding Buffer
ii) 3ml dI/dC
iii) Q to 20ml (see table)
iv) competitor at 10-20X excess
v) 10-50 fold dilution of the probe
vi) NE usually 2-4mg 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.