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

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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
141.0 μL--3.0 μL
251.0 μL-2.0 μL1.0 μL
361.0 μL2.0 μL-1.0 μL
471.0 μL2.0 μL1.0 μL-
581.0 μL1.0 μL2.0 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 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[[:Category:{{{1}}}|{{{1}}}]] (tank in refrigerator, power supply outside at room temperature)

Results of the gel are on the July 12 page

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