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Summary: Auble Lab
Gel Shift Assay for Detection of TBP-DNA Complexes and Mot1 activity
6% Polyacrylamide Gel (14 cm X 16 cm X 0.2 cm)
Stock acrylamide (20% acrylamide/0.33% bis acryl) 10.5 ml
10 X TG buffer 3.5 ml
50% glycerol 1.75 ml
0.5 M DTT 35.0 µl
1 M magnesium acetate 0.35 ml
water 18.9 ml
35.0 ml total
de-gas, then add 0.3 ml 10% APS and 30 µl TEMED
Running buffer is 1 X TG buffer with 5 mM magnesium acetate
(60 ml 10 X buffer + 3 ml 1 M magnesium acetate + water: 600 ml final volume)
10 X TG buffer (500 mls)
15.0 g Tris
71.4 g glycine
1.9 g disodium EDTA
adjust pH to 8.3
Pre-run gel at 100 V in cold room while setting up the binding reactions.
5 X Binding Buffer (store at -20 C)
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