EMSA using ds Oligonucleotides【Harvard University】

Cepko/Tabin Lab ,Harvard University http://axon.med.harvard.edu/~cepko/protocol/mike/E1.html Cepko/Tabin Lab ,Harvard University http://axon.med.harvard.edu/~cepko/protocol/mike/E1.html Solutions

10X Annealing Buffer

200 mM Tris 8.0 200 ml 1M Tris pH 8.0 ml .5M EDTA pH 8.0 ml 5M NaCl ml Q store at room temperature

10X Klenow Buffer

500 mM Tris 7.5 500

100 mM MgCl₂

ml 1M Tris pH 7.5 2 100 ml 1M MgCl₂

10 mM DTT 20

0.5

330

ml 0.5 M DTT m g/ ml BSA 50 ml 10 mg/ml BSA (NEB)ml Q store at -80℃ in 50 ml aliq.

2X Binding Buffer (ref: Gutman et al.GENE 110,1992 pg 197)

20% glycerol 400

20 mM Tris 7.5 20

100 mM KCl 100

1 mM DTT 2

478

ml 50% glycerol ml 1M Tris pH 7.5 ml 1M KCl ml 0.5 M DTT ml Q make fresh as needed

Poly dI/dC

Make a 1

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

• Mix the following and incubate at room temperature for 30 min:

1

5

25

dGTP)

5

14

1

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

• 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

Cool to 4℃ along with the appropriate amount of 1X TBE.

• Mix the binding reagents in the following order:

i)10

ii)3

iii)Q to 20

iv)competitor at 10-20X excess