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° C 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
