IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-7-11: Difference between revisions
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* stored in a microcentrifuge tube in the "Shawn nanotube supplies" box at 4{{c}} | * stored in a microcentrifuge tube in the "Shawn nanotube supplies" box at 4{{c}} | ||
Thrombin-aptamer binding experiment: | Loading dye | ||
* 100 {{ul}} 5x Bock selection buffer, 400 {{ul}} water, 500 {{ul}} 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 [[http://openwetware.org/wiki/IGEM:Harvard/2006/DNA_nanostructures/Protocols|Dr. Shih's assay]]): | |||
* General info: | * General info: | ||
Line 25: | Line 29: | ||
** Final volume of each: 4 {{ul}} | ** Final volume of each: 4 {{ul}} | ||
** 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) | ** 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: | |||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Tube''' | | align="center" style="background:#f0f0f0;"|'''Tube''' | ||
| align="center" style="background:#f0f0f0;"|'''Lane''' | |||
| align="center" style="background:#f0f0f0;"|'''Bock's selection buffer''' (4x) | | align="center" style="background:#f0f0f0;"|'''Bock's selection buffer''' (4x) | ||
| align="center" style="background:#f0f0f0;"|'''Aptamer''' (2 {{um}}) | | align="center" style="background:#f0f0f0;"|'''Aptamer''' (2 {{um}}) | ||
Line 33: | Line 38: | ||
| align="center" style="background:#f0f0f0;"|'''dH<sub>2</sub>O''' | | align="center" style="background:#f0f0f0;"|'''dH<sub>2</sub>O''' | ||
|- | |- | ||
| 1||1.0 {{ul}}||-||-||3.0 {{ul}} | | 1||4||1.0 {{ul}}||-||-||3.0 {{ul}} | ||
|- | |- | ||
| 2||1.0 {{ul}}||-||2.0 {{ul}}||1.0 {{ul}} | | 2||5||1.0 {{ul}}||-||2.0 {{ul}}||1.0 {{ul}} | ||
|- | |- | ||
| 3||1.0 {{ul}}||2.0 {{ul}}||-||1.0 {{ul}} | | 3||6||1.0 {{ul}}||2.0 {{ul}}||-||1.0 {{ul}} | ||
|- | |- | ||
| 4||1.0 {{ul}}||2.0 {{ul}}||1.0 {{ul}}||- | | 4||7||1.0 {{ul}}||2.0 {{ul}}||1.0 {{ul}}||- | ||
|- | |- | ||
| 5||1.0 {{ul}}||1.0 {{ul}}||2.0 {{ul}}||- | | 5||8||1.0 {{ul}}||1.0 {{ul}}||2.0 {{ul}}||- | ||
|} | |} | ||
* 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{{c}} | |||
** 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{{c}} (tank in refrigerator, power supply outside at room temperature) | |||
Results of the gel are on the [[IGEM:Harvard/2006/DNA_nanostructures/Notebook/2006-7-12|July 12]] page |
Latest revision as of 15:42, 13 July 2006
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 | 4 | 1.0 μL | - | - | 3.0 μL |
2 | 5 | 1.0 μL | - | 2.0 μL | 1.0 μL |
3 | 6 | 1.0 μL | 2.0 μL | - | 1.0 μL |
4 | 7 | 1.0 μL | 2.0 μL | 1.0 μL | - |
5 | 8 | 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 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