IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-7-13: Difference between revisions
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2. Protocol | 2. Protocol | ||
- gel run @ 120V for 1.25 hrs. | - 12% Invitrogen polyacrylamide gel run @ 120V for 1.25 hrs. | ||
- gel cut into DNA & protein sections after removal from cartridge (slight tearing towards bottom) | - gel cut into DNA & protein sections after removal from cartridge (slight tearing towards bottom) | ||
- protein section stained w/ GelCode Blue Stain (Coomassie Blue) for 1 hr. | - protein section stained w/ GelCode Blue Stain (Coomassie Blue) for 1 hr. | ||
Revision as of 15:41, 13 July 2006
Container 3.2
Initial Container v3.2 Preparation and Analysis
- Oligos arrived this morning from Invitrogen. Plan for today is to mix oligos together into pre-stocks, and then working stocks, and then try some initial folding experiments and gel analysis.
Creating pre-working stocks and working stocks from design 3.2 oligos
- Mix 10 μL of each 50 μM stock oligo
| oligo | desc | plate location | total |
| c3.2.1 | core barrel | 3.2p1A01–3.2p1H10 | 94 |
| c3.2.2 | core top lid | 3.2p2A01–3.2p2C04 | 28 |
| c3.2.3 | core bottom lid | 3.2p2C05–3.2p2E10 | 30 |
| c3.2.4 | barrel at inside apatamer locations -aptamers | 3.2p2E11–3.2p2F01 | 3 |
| c3.2.5 | barrel at outside aptamer locations -aptamers | 3.2p2F02–3.2p2F04 | 3 |
| c3.2.6 | barrel at inside apatamer locations +aptamers | 3.2p2F05–3.2p2F07 | 3 |
| c3.2.7 | barrel at outside aptamer locations +aptamers | 3.2p2F08–3.2p2F10 | 3 |
| c3.2.8 | barrel at latch locations -latches | 3.2p2F11–3.2p2F12 | 2 |
| c3.2.9 | lid at latch locations -latches (empty) | 0 | |
| c3.2.10 | barrel at latch locations +latch1 +latch2 | 3.2p2G01–3.2p2G02 | 2 |
| c3.2.11 | latch from barrel +latch1 -latch2 | 3.2p2G03–3.2p2G04 | 2 |
| c3.2.12 | latch from barrel -latch1 +latch2 | 3.2p2G05–3.2p2G06 | 2 |
| c3.2.13 | lid at latch locations +latch1 +latch2 (empty) | 0 | |
| c3.2.14 | latch from lids +latch1 -latch2 | 3.2p2G07–3.2p2G08 | 2 |
| c3.2.15 | latch from lids -latch1 +latch2 | 3.2p2G09–3.2p2G10 | 2 |
| c3.2.16 | latch staples +latch2 | 3.2p2G11–3.2p2H02 | 4 |
| c3.2.17 | displacement strands latch1 | 3.2p2H03–3.2p2H06 | 4 |
| c3.2.18 | displacement strands latch2 | 3.2p2H07–3.2p2H10 | 4 |
Make c3.2 Working stocks
c3.2 Folding Experiment
New PCR routine
- on PTC-100 machine, called FOLDINGD
- step 1: 80[[:Category:{{{1}}}|{{{1}}}]] for 2 min, increment -1.0[[:Category:{{{1}}}|{{{1}}}]] each cycle
- step 2: go to step 1, 59 more times
- step 3: end
Gel analysis
Protein & DNA-staining Experiment (Repeat 1)
1. Rxn Mixtures
| 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 | 8 | 0.65 | 0.5 | 6.5 | 3.0 | 10.0 |
| 2 | 9 | 1.30 | 1.0 | 6.0 | 3.0 | 10.0 |
| 3 | 10 | 1.95 | 1.5 | 5.5 | 3.0 | 10.0 |
| 4 | 11 | 2.69 | 2.0 | 5.0 | 3.0 | 10.0 |
| Tube | Lane | 2 μM Aptamer (μL) | water (μL) | 10x loading dye (μL) | total volume (μL) |
| 1 | 2 | 1.0 | 6.0 | 3.0 | 10.0 |
| 2 | 3 | 2.0 | 5.0 | 3.0 | 10.0 |
| 3 | 4 | 3.0 | 4.0 | 3.0 | 10.0 |
| 4 | 5 | 4.0 | 3.0 | 3.0 | 10.0 |
2. Protocol
- 12% Invitrogen polyacrylamide gel run @ 120V for 1.25 hrs. - gel cut into DNA & protein sections after removal from cartridge (slight tearing towards bottom) - protein section stained w/ GelCode Blue Stain (Coomassie Blue) for 1 hr. - DNA section stained w/ 10μL EtBr in 100 mL Tris-glycine buffer for 1 hr.
3. Results
- loading dye moved about 2/3 of the way down the gel - protein did not image - small spot of DNA in lane 4
Protein & DNA-staining Experiment (Repeat 2)
1. Rxn Mixtures
| Tube | Lane | Thrombin mass (μg) | 2 μM thrombin (μL) (2 μM = 1.299 μg/μL) |
water (μL) | 10x loading dye (μL) | total volume (μL) |
| 0 | 1 | 0 | 0.0 | 7.0 | 3.0 | 10.0 |
| 1 | 2 | 0.65 | 0.5 | 6.5 | 3.0 | 10.0 |
| 2 | 3 | 1.30 | 1.0 | 6.0 | 3.0 | 10.0 |
| 3 | 4 | 1.95 | 1.5 | 5.5 | 3.0 | 10.0 |
| 4 | 5 | 2.69 | 2.0 | 5.0 | 3.0 | 10.0 |
| Tube | Lane | 2 μM Aptamer (μL) | water (μL) | 10x loading dye (μL) | total volume (μL) |
| 0 | 7 | 0.0 | 7.0 | 3.0 | 10.0 |
| 1 | 8 | 1.0 | 6.0 | 3.0 | 10.0 |
| 2 | 9 | 2.0 | 5.0 | 3.0 | 10.0 |
| 3 | 10 | 3.0 | 4.0 | 3.0 | 10.0 |
| 4 | 11 | 4.0 | 3.0 | 3.0 | 10.0 |
2. Protocol
- 12% Invitrogen polyacrylamide gel run @ 120V for 15 min. - gel cut into DNA & protein sections after removal from cartridge (slight tearing towards bottom) - protein section stained w/ GelCode Blue Stain (Coomassie Blue) (the same Coomassie Blue used in repeat 1) for 30 min. - DNA section stained w/ 10μL EtBr in 100 mL Tris-glycine buffer for 30 min.
3. Results
- both protein and DNA successfully imaged - protein appears to be in distinct bands, implying that there are several different sized molecules in the protein mixture
