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Protocol E

Protocol E.1

EMSA using ds Oligonucleotides

 

Solutions

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 MgCl2 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.