IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-7-12: Difference between revisions
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* gel catridge opened and cut between protein and DNA sections | * gel catridge opened and cut between protein and DNA sections | ||
** was cut before removal from one side of cartridge and led to some tearing during removal from that side, cut after removal next time | ** was cut before removal from one side of cartridge and led to some tearing during removal from that side, cut after removal next time | ||
* protein section was rocked in GelCode Blue Stain for | * protein section was rocked in GelCode Blue Stain for 1 hr. | ||
* DNA section was rocked in 100 mL Tris-glycine buffer with 10 {{ul}} of 10 mg/mL ethidium bromide for | * DNA section was rocked in 100 mL Tris-glycine buffer with 10 {{ul}} of 10 mg/mL ethidium bromide for 1 hr. | ||
Revision as of 12:28, 12 July 2006
Results from 7/11 Thrombin-Binding Experiment
- gels removed at 10:00 am: appear to have run off the gel (very hard to tell since they were so faint to begin with)
- removed gels from plastic
- cut bottom of gel where it sticks out of narrow hole at the bottom
- pried two plastic sides apart (gel now stuck to one side)
- loosen top edge by sliding flat tool under it a bit.
- make sure the very bottom is completely cut
- run it under water over the a plastic tupperware container - move it back and forth left to right - eventually the gel should come free into the container.
- hold the gel in the container while you pour the water out.
- stained with GelCode Blue Stain (Coomassie blue) (in brown bottle in first 4[[:Category:{{{1}}}|{{{1}}}]] fridge)
- pour enough Coomassie blue over it to cover the gel.
- put the lid on the tupperware and put it on the rocker for about 20 minutes.
Protein- and DNA-staining control experiment
- goal: to load varying concentrations of thrombin onto a gel and stain with Coomassie blue in an attempt to image the protein, and various concentrations of DNA onto a gel and stain with a yet-to-be determined stain to image the DNA
| Tube | Lane | Thrombin mass (μg) | 2 μM thrombin (μL) (2 μM = 1.299 μg/μL) |
water (μL) | 10x loading dye (μL) | total volume (μL) |
| 1 | 1 | 0.65 | 0.5 | 8.5 | 2.0 | 11.0 |
| 2 | 2 | 1.3 | 1.0 | 8.0 | 2.0 | 11.0 |
| 3 | 3 | 2.6 | 2.0 | 7.0 | 2.0 | 11.0 |
| 4 | 4 | 3.9 | 3.0 | 6.0 | 2.0 | 11.0 |
| 5 | 5 | 5.2 | 4.0 | 5.0 | 2.0 | 11.0 |
| Tube | Lane | 2 μM Aptamer (μL) | water (μL) | 10x loading dye (μL) | total volume (μL) |
| 1 | 7 | 1.0 | 8.0 | 3.0 | 12.0 |
| 2 | 8 | 2.0 | 7.0 | 3.0 | 12.0 |
| 3 | 9 | 3.0 | 6.0 | 3.0 | 12.0 |
| 4 | 10 | 4.0 | 5.0 | 3.0 | 12.0 |
| 5 | 11 | 5.0 | 4.0 | 3.0 | 12.0 |
- all reagents were mixed in individual 0.2 mL PCR tubes
- run at 25 V for 2.5 h.
- loading dye appears to have diffused about 3 mm in all directions into the gel
- gel catridge opened and cut between protein and DNA sections
- was cut before removal from one side of cartridge and led to some tearing during removal from that side, cut after removal next time
- protein section was rocked in GelCode Blue Stain for 1 hr.
- DNA section was rocked in 100 mL Tris-glycine buffer with 10 μL of 10 mg/mL ethidium bromide for 1 hr.
