EMSA using ds Oligonucleotides

 

EMSA using ds Oligonucleotides

 

 

Solutions

10X Annealing Buffer

200 mM Tris 8.0 200 m l 1M Tris pH 8.0

10 mM EDTA 8.0 20 m l .5M EDTA pH 8.0

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° C 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° 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 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° C along with the appropriate amount of 1X TBE.

• Mix the binding reagents in the following order:

i) 10 m l 2X Binding Buffer

ii) 3 m l dI/dC

iii) Q to 20 m l (see table)

iv) competitor at 10-20X excess

v) 10-50 fold dilution of the probe

vi) 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.