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 7.5 500
ml 1M Tris pH 7.5
100 mM MgCl
2 100 ml 1M MgCl2
10 mM DTT 20
ml 0.5 M DTT
0.5
mg/ml BSA 50 ml 10 mg/ml BSA (NEB)
330
ml Q
store at -80° C in 50 ml aliq.
2X Binding Buffer (ref: Gutman et al. GENE 110, 1992 pg 197)
20% glycerol 400
ml 50% glycerol
20 mM Tris 7.5 20
ml 1M Tris pH 7.5
100 mM KCl 100
ml 1M KCl
1 mM DTT 2
ml 0.5 M DTT
478
ml Q
make fresh as needed
Poly dI/dC
Make a 1
mg/ml stock and store in 100 ul aliq. at -80° C
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 ml 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:
1 ml of the annealed oligo mixture
5 ml 10X Klenow buffer
25 ml dNTP mix (21 ml Q, 3 ml 10 mM dATP/dTTP
dGTP)
5 ml a32P dCTP
14 ml Q
1 ml 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 ml 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.
EMSA Gel:
8 ml 30% acrylamide (0.8% BIS)
6 ml 50% glycerol
6 ml 10X TBE
40 ml Q
180 ml 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) 10 ml 2X Binding Buffer
ii) 3 ml dI/dC
iii) Q to 20 ml (see table)
iv) competitor at 10-20X excess
v) 10-50 fold dilution of the probe
vi) NE usually 2-4 mg 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.
FOOTPRINTING WITH DNASE1