IGEM:Harvard/2006/DNA/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}}}]]

Thrombin-aptamer binding experiment:

• 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)
• 1:
  • 1.0 μL of 4x (not 5x) Bock's selection buffer
  • 3.0 μL water
• 2:
  • 1.0 μL of 4x (not 5x) Bock's selection buffer
  • 2.0 μL of 2 μM thrombin (final concentration: 1.0 μM = 4 pmol)
  • 1.0 μL water
• 3:
  • 1.0 μL of 4x (not 5x) Bock's selection buffer
  • 2.0 μL of 2 μM aptamers (final concentration: 1.0 μM = 4 pmol)
  • 1.0 μL water
• 4:
  • 1.0 μL of 4x (not 5x) Bock's selection buffer
  • 2.0 μL of 2 μM aptamers (final concentration: 1.0 μM = 4 pmol)
  • 1.0 μL of 2 μM thrombin (final concentration: 0.5 μM = 2 pmol)
• 5:
  • 1.0 μL of 4x (not 5x) Bock's selection buffer
  • 1.0 μL of 2 μM aptamers (final concentration: 0.5 μM = 2 pmol)
  • 2.0 μL of 2 μM thrombin (final concentration: 1.0 μM = 4 pmol)