IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-7-11

Reconstituted lyophilized bovine thrombin
 * biuret is 745 NIH units = 637 (1170 NIH units = 1 mg)
 * "A suggested concentration for preparation of a stock solution is 100 units/ml. The solution should contain approximately 0.1% BSA for stability and is stable for about one week at 0-5 °C. Since thrombin solutions adsorb to glass, it is recommended to aliquot the solution in plastic tubes and store at -20 °C or below."
 * Made a stock solution: reconstituted 745 NIH units in 0.490 mL of 0.1% BSA to give a working stock of 1520 units / mL = 1299 / mL = 20 nmol / mL = 20  (formula weight is approximately 65 kDa )
 * 0.1% BSA = 20 mg BSA in total volume of 20 mL water
 * stock solution stored in one of the blue benchtop coolers in the door of the -20 freezer

Diluted thrombin stock
 * 5 of 20  thrombin stock and 45  0.1% BSA to make 2  stock
 * briefly vortexed protein / BSA sol'n mixture (perhaps a poor idea? Matthewmeisel)
 * stored in one of the blue benchtop coolers in the door of the -20 freezer

Diluted 5x Bock's selection buffer
 * 1,000 selection buffer and 250  water to make 4x stock
 * stored in a microcentrifuge tube with the other buffer bottles

Diluted 6hbah1 aptamer
 * 2 of 100  aptamer stock and 98  water to make 2  stock
 * stored in a microcentrifuge tube in the "Shawn nanotube supplies" box at 4

Loading dye
 * 100 5x Bock selection buffer, 400  water, 500  glycerol, small spatula-tip of bromthymol blue (sodium salt) to make 10x stock
 * stored in a microcentrifuge tube with the other loading dyes by the electrophoresis tanks in the large lab room

Thrombin-aptamer binding experiment (based on [|Dr. Shih's assay]):


 * General info:
 * All ingredients pipetted into respective 0.2 mL PCR tubes
 * Final volume of each: 4
 * Used aptamer 6hbab1 (5'-AGGATCCCCGGGTACCGGCTAGTACCCGTATAGGTTGGTGTGGTTGG-3'), which binds to a standard 6-helix-bundle nanotube (5' end) and contains a thrombin aptamer sequence (3' end)
 * Ingredients:
 * Incubated at room temperature for 30 min. (Turned out to be closer to 45 min.)
 * Added 5 mL 1x Bock's selection buffer and 1 mL 10x loading dye
 * Loaded onto a non-denaturing polyacrylamide gel (10% to 20% gradient) at 4
 * Lanes 1-3: loading dye (practice)
 * Lanes 4-8: tubes 1-5, respectively
 * Lanes 9-10: empty
 * Lanes 11-12: Lewis' experiment
 * ran 10-20% Invitrogen polyacrylamide gel at 15 V starting at 4:15 pm at 4 (tank in refrigerator, power supply outside at room temperature)

Results of the gel are on the July 12 page