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 |
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