IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-7-13: Difference between revisions
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====Initial Container v3.2 Preparation and Analysis==== | ====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. | *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. | ||
*Oligos were ''very'' frozen and were left overnight at 4{{c}} to thaw. | |||
====New folding conditions==== | |||
* on PTC-100 machine, called FOLDINGD | * on PTC-100 machine, called FOLDINGD | ||
* step 1: 80{{c}} for 2 min, increment -1.0{{c}} each cycle | * step 1: 80{{c}} for 2 min, increment -1.0{{c}} each cycle | ||
| Line 61: | Line 12: | ||
* step 3: end | * step 3: end | ||
==== | ====Ordered 3'-biotinylated oligos==== | ||
*...because we expect biotin-streptavidin binding to be more reliable than thrombin-aptamer binding | |||
c.3.2.6.1b GGAATAGGGAACCTATTATTCACCCTCAGAGCCACTTTCATCTTT-biotin | |||
c.3.2.6.2b AAGCACTAGTAAAAGAGTCTGACTTGCCTGAGTAGACAGAGGTTT-biotin | |||
c.3.2.6.3b AGTCAGAAGCAAAGCGGATTGGTAATAGTAAAATGTTT-biotin | |||
c.3.2.7.1b GGCAAAAATCAGCTTGCTTTCTTTCAACAGTTTCAATAGCCCTTT-biotin | |||
c.3.2.7.2b TCAGATGATGGCAATTCATCACACCTTGCTGAACCTTT-biotin | |||
c.3.2.7.3b TTTATCCTCTTTCCAGAGCCTTTT-biotin | |||
==Protein & DNA-staining Experiment (Repeat 1)== | ==Protein & DNA-staining Experiment (Repeat 1)== | ||
| Line 102: | Line 61: | ||
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. | ||
| Line 154: | Line 113: | ||
2. Protocol | 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/ 10{{ul}} EtBr in 100 mL Tris-glycine buffer for 30 min. | |||
3. Results | [[Image:20060712_thrombin.jpg|thumb|Thrombin, 12% PAGE]][[Image:20060712_aptamers.jpg|thumb|ss DNA, 12% PAGE]]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 | |||
*perhaps the fastest band is thrombin monomer, the second fastest is dimer, etc. | |||
Latest revision as of 19:24, 18 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.
- Oligos were very frozen and were left overnight at 4[[:Category:{{{1}}}|{{{1}}}]] to thaw.
New folding conditions
- 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
Ordered 3'-biotinylated oligos
- ...because we expect biotin-streptavidin binding to be more reliable than thrombin-aptamer binding
c.3.2.6.1b GGAATAGGGAACCTATTATTCACCCTCAGAGCCACTTTCATCTTT-biotin c.3.2.6.2b AAGCACTAGTAAAAGAGTCTGACTTGCCTGAGTAGACAGAGGTTT-biotin c.3.2.6.3b AGTCAGAAGCAAAGCGGATTGGTAATAGTAAAATGTTT-biotin c.3.2.7.1b GGCAAAAATCAGCTTGCTTTCTTTCAACAGTTTCAATAGCCCTTT-biotin c.3.2.7.2b TCAGATGATGGCAATTCATCACACCTTGCTGAACCTTT-biotin c.3.2.7.3b TTTATCCTCTTTCCAGAGCCTTTT-biotin
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
- perhaps the fastest band is thrombin monomer, the second fastest is dimer, etc.
