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 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 (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:

Incubated at room temperature for 30 min.
Loadd onto a non-denaturing polyacrylamide gel (10% to 20% gradient) at 4[[:Category:{{{1}}}|{{{1}}}]]
Ran at 130 V for approximately 90 min at 4[[:Category:{{{1}}}|{{{1}}}]]

Navigation menu