Matt Gethers/CRI, Thailand/Labwork/Binding Assay

=Protein DNA Binding Assay using Fluorescence Anisotropy=


 * 1) Anneal BT2767 and BT2768 to form the HmgA promter probe and anneal BT2769 and BT2770 to form the Gpx1 promoter probe. Add 10 &mu;l of each primer to 80 &mu;l of water, boil at 100 oC for 5 minutes, then turn off heat block and allow to cool to room temperature.
 * 2) Prepare binding reaction buffer (see below).
 * 3) Prepare dilutions of peptide. Aim for 20, 10, 5, 2, and 1 &mu;M. Do serial dilutions. See the table for a suggested dilution pattern.
 * 4) Place dilutions on ice and get polydIdC and 1 M DTT.
 * 5) Get cuvettes from the room with the FA equipment. Add 900 &mu;l water, 100 &mu;l 10x buffer, 1 &mu;l 0.5 mg/ml polydIdC, and 1 &mu;l 1 M DTT.
 * 6) Mix the contents of the cuvette, clean with a kim wipe, and place in the reader to measure intensity and basal anisotropy.
 * 7) Begin by adding 1 &mu;l samples of 1 &mu;M dilution to the cuvette. Mix and place in reader. Continue by adding more of 1 &mu;M, then move on to 2, 5, 10, and 20.

Dilution Table

 * Note: Because you're adding different volumes of buffer to each dilution, you can't make a stock of a particular concentration and use in each. Make up a stock of 10x buffer with DTT (16.3 &mu;l 10x buffer and 1.63 &mu;l 100 mM DTT will make enough for the 5 dilutions outlined in the table), then add different amounts of this stock and water to each dilution. Table follows:

Run Notes
8.7.08 Run Notes

8.8.08 Run Notes