User:Matthewmeisel/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
- stored in one of the blue benchtop coolers in the door of the -20[[:Category:{{{1}}}|{{{1}}}]] freezer
Thrombin-aptamer binding experiments (4 μL each)
- Experiment 1:
- 1.0 μL of 4x (not 5x) Bock's selection buffer
- 3.0 μL water
- Experiment 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
- Experiment 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
- Experiment 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)
- Experiment 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)