IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-17: Difference between revisions
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* conduct 2nd PEG precipitation on yesterday's precipitated samples (5.0A, C, D 7%-10%) | * conduct 2nd PEG precipitation on yesterday's precipitated samples (5.0A, C, D 7%-10%) | ||
* protocol: mix all ingredients, add nanostructures last, total volume is 100 mL, incubate on ice for 15 min, spin for 10 min at 16k rcf, pipet off supernatant, resuspend pellet in 100 {{ul}} water | * protocol: mix all ingredients, add nanostructures last, total volume is 100 mL, incubate on ice for 15 min, spin for 10 min at 16k rcf, pipet off supernatant, resuspend pellet in 100 {{ul}} water | ||
* CONCLUSIONS: we seem to be unable to perform 2 PEG precipitations because all of the nanostructures ended up in the supernatant. we will repeat the 1st PEG precipitation with c5.0 A, C, D at 6% and probably not repeat another PEG if the protection assay is okay. | |||
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==Magnetic Streptavidin Protection, Take 3== | ==Magnetic Streptavidin Protection, Take 3== | ||
Revision as of 15:32, 17 August 2006
2nd PEG Precipitation
- conduct 2nd PEG precipitation on yesterday's precipitated samples (5.0A, C, D 7%-10%)
- protocol: mix all ingredients, add nanostructures last, total volume is 100 mL, incubate on ice for 15 min, spin for 10 min at 16k rcf, pipet off supernatant, resuspend pellet in 100 μL water
- CONCLUSIONS: we seem to be unable to perform 2 PEG precipitations because all of the nanostructures ended up in the supernatant. we will repeat the 1st PEG precipitation with c5.0 A, C, D at 6% and probably not repeat another PEG if the protection assay is okay.
| Final PEG % | PEG μL | NaCl μL | c5.0 μL | dH2O μL |
| 7 | 35 | 10 | 40 | 15 |
| 8 | 40 | 10 | 40 | 10 |
| 9 | 45 | 10 | 40 | 5 |
| 10 | 50 | 10 | 40 | 0 |
| lane | contents | loading dye |
| 1 | 8 μL untreated 5.0.A | 3 μL |
| 2 | 20 μL 5.0.A.1 in 7% PEG and 0.5 M NaCl (supernatant) | 3 μL |
| 3 | 8 μL 5.0.A.1 in 7% PEG and 0.5 M NaCl (pellet) | 3 μL |
| 4 | 20 μL 5.0.A.2 in 7% PEG and 0.5 M NaCl (supernatant) | 3 μL |
| 5 | 8 μL 5.0.A.2 in 7% PEG and 0.5 M NaCl (pellet) | 3 μL |
| 6 | 20 μL 5.0.A.1 in 8% PEG and 0.5 M NaCl (supernatant) | 3 μL |
| 7 | 8 μL 5.0.A.1 in 8% PEG and 0.5 M NaCl (pellet) | 3 μL |
| 8 | 20 μL 5.0.A.2 in 8% PEG and 0.5 M NaCl (supernatant) | 3 μL |
| 9 | 8 μL 5.0.A.2 in 8% PEG and 0.5 M NaCl (pellet) | 3 μL |
| 10 | 1 kb+ ladder | 3 μL |
| 11 | 2.7 μL p7308 | 3 μL |
| 12 | 20 μL 5.0.A.1 in 9% PEG and 0.5 M NaCl (supernatant) | 3 μL |
| 13 | 8 μL 5.0.A.1 in 9% PEG and 0.5 M NaCl (pellet) | 3 μL |
| 14 | 20 μL 5.0.A.2 in 9% PEG and 0.5 M NaCl (supernatant) | 3 μL |
| 15 | 8 μL 5.0.A.2 in 9% PEG and 0.5 M NaCl (pellet) | 3 μL |
| 16 | 20 μL 5.0.A.1 in 10% PEG and 0.5 M NaCl (supernatant) | 3 μL |
| 17 | 8 μL 5.0.A.1 in 10% PEG and 0.5 M NaCl (pellet) | 3 μL |
| 18 | 20 μL 5.0.A.2 in 10% PEG and 0.5 M NaCl (supernatant) | 3 μL |
| 19 | 8 μL 5.0.A.2 in 10% PEG and 0.5 M NaCl (pellet) | 3 μL |
| 20 | empty | - |
| 21 | 8 μL untreated 5.0.C | 3 μL |
| 22 | 20 μL 5.0.C.1 in 7% PEG and 0.5 M NaCl (supernatant) | 3 μL |
| 23 | 8 μL 5.0.C.1 in 7% PEG and 0.5 M NaCl (pellet) | 3 μL |
| 24 | 20 μL 5.0.C.2 in 7% PEG and 0.5 M NaCl (supernatant) | 3 μL |
| 23 | 8 μL 5.0.C.2 in 7% PEG and 0.5 M NaCl (pellet) | 3 μL |
| 24 | 20 μL 5.0.C.1 in 8% PEG and 0.5 M NaCl (supernatant) | 3 μL |
| 25 | 8 μL 5.0.C.1 in 8% PEG and 0.5 M NaCl (pellet) | 3 μL |
| 26 | 20 μL 5.0.C.2 in 8% PEG and 0.5 M NaCl (supernatant) | 3 μL |
| 27 | 8 μL 5.0.C.2 in 8% PEG and 0.5 M NaCl (pellet) | 3 μL |
| 28 | 20 μL 5.0.C.1 in 9% PEG and 0.5 M NaCl (supernatant) | 3 μL |
| 29 | 8 μL 5.0.C.1 in 9% PEG and 0.5 M NaCl (pellet) | 3 μL |
| 30 | 1 kb+ ladder | 3 μL |
| 31 | 2.7 μL p7308 | 3 μL |
| 32 | 20 μL 5.0.C.2 in 9% PEG and 0.5 M NaCl (supernatant) | 3 μL |
| 33 | 8 μL 5.0.C.2 in 9% PEG and 0.5 M NaCl (pellet) | 3 μL |
| 34 | 20 μL 5.0.C.1 in 10% PEG and 0.5 M NaCl (supernatant) | 3 μL |
| 35 | 8 μL 5.0.C.1 in 10% PEG and 0.5 M NaCl (pellet) | 3 μL |
| 36 | 20 μL 5.0.C.2 in 10% PEG and 0.5 M NaCl (supernatant) | 3 μL |
| 37 | 8 μL 5.0.C.2 in 10% PEG and 0.5 M NaCl (pellet) | 3 μL |
| 38-40 | empty | - |
| 41 | 8 μL untreated 5.0.D | 3 μL |
| 42 | 20 μL 5.0.D.1 in 7% PEG and 0.5 M NaCl (supernatant) | 3 μL |
| 43 | 8 μL 5.0.D.1 in 7% PEG and 0.5 M NaCl (pellet) | 3 μL |
| 44 | 20 μL 5.0.D.2 in 7% PEG and 0.5 M NaCl (supernatant) | 3 μL |
| 45 | 8 μL 5.0.D.2 in 7% PEG and 0.5 M NaCl (pellet) | 3 μL |
| 46 | 20 μL 5.0.D.1 in 8% PEG and 0.5 M NaCl (supernatant) | 3 μL |
| 47 | 8 μL 5.0.D.1 in 8% PEG and 0.5 M NaCl (pellet) | 3 μL |
| 48 | 20 μL 5.0.D.2 in 8% PEG and 0.5 M NaCl (supernatant) | 3 μL |
| 49 | 8 μL 5.0.D.2 in 8% PEG and 0.5 M NaCl (pellet) | 3 μL |
| 50 | 1 kb+ ladder | 3 μL |
| 51 | 2.7 μL p7308 | 3 μL |
| 52 | 20 μL 5.0.D.1 in 9% PEG and 0.5 M NaCl (supernatant) | 3 μL |
| 53 | 8 μL 5.0.D.1 in 9% PEG and 0.5 M NaCl (pellet) | 3 μL |
| 54 | 20 μL 5.0.D.2 in 9% PEG and 0.5 M NaCl (supernatant) | 3 μL |
| 55 | 8 μL 5.0.D.2 in 9% PEG and 0.5 M NaCl (pellet) | 3 μL |
| 56 | 20 μL 5.0.D.1 in 10% PEG and 0.5 M NaCl (supernatant) | 3 μL |
| 57 | 8 μL 5.0.D.1 in 10% PEG and 0.5 M NaCl (pellet) | 3 μL |
| 58 | 20 μL 5.0.D.2 in 10% PEG and 0.5 M NaCl (supernatant) | 3 μL |
| 59 | 8 μL 5.0.D.2 in 10% PEG and 0.5 M NaCl (pellet) | 3 μL |
| 60 | empty | - |
Magnetic Streptavidin Protection, Take 3
- Performed steps 9-12, as in Wed's protocol.
- Gel:
| Lane | Component | Amount |
| 1 | 1kb ladder | 10uL |
| 2 | p7308 (44nM) | 7.5uL |
| 3 | c5.0.D wash | 40uL |
| 4 | c5.0.D final | 40uL |
| 5 | c5.0.8(b) wash | 40uL |
| 6 | c5.0.8(b) final | 40uL |
| 7 | c5.0.Eb wash | 40uL |
| 8 | c5.0.Eb final | 40uL |
| 9 | c5.0.Fb wash | 40uL |
| 10 | c5.0.Fb final | 40uL |
