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

Reconstituted lyophilized bovine thrombin

• biuret is 745 NIH units = 637 μg (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." [1]
• 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 μg / mL = 20 nmol / mL = 20 μM (formula weight is approximately 65 kDa [2])
  • 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[[:Category:{{{1}}}|{{{1}}}]] freezer

Diluted thrombin stock

• 5 μL of 20 μM thrombin stock and 45 μL 0.1% BSA to make 2 μM 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[[:Category:{{{1}}}|{{{1}}}]] freezer

Diluted 5x Bock's selection buffer

• 1,000 μL selection buffer and 250 μL water to make 4x stock
• stored in a microcentrifuge tube with the other buffer bottles

Diluted 6hbah1 aptamer

• 2 μL of 100 μM aptamer stock and 98 μL water to make 2 μM stock
• stored in a microcentrifuge tube in the "Shawn nanotube supplies" box at 4[[:Category:{{{1}}}|{{{1}}}]]

Loading dye

• 100 μL 5x Bock selection buffer, 400 μL water, 500 μL 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 [Shih's assay]):

• General info:
  • All ingredients pipetted into respective 0.2 mL PCR tubes
  • Final volume of each: 4 μL
  • 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:

Tube	Lane	Bock's selection buffer (4x)	Aptamer (2 μM)	Thrombin (2 μM)	dH2O
1	3	1.0 μL	-	-	3.0 μL
2	4	1.0 μL	-	2.0 μL	1.0 μL
3	5	1.0 μL	2.0 μL	-	1.0 μL
4	6	1.0 μL	2.0 μL	1.0 μL	-
5	7	1.0 μL	1.0 μL	2.0 μL	-

• 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[[:Category:{{{1}}}|{{{1}}}]]
  • Lanes 0-2: loading dye (practice)
  • Lanes 3-7: tubes 1-5, respectively
  • Lanes 8-9: empty
  • Lanes 10-11: Lewis' experiment
• Ran at 15 V starting at 4:15 pm at 4[[:Category:{{{1}}}|{{{1}}}]] (tank in refrigerator, power supply outside at room temperature)

Results of the gel are on the July 12 page