IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-16: Difference between revisions
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==Protection assay== | |||
Revision as of 12:06, 16 August 2006
Repeat PEG Precipitation + Protection Assay
Overview
- concentrate on 30 mM MgCl2 folding buffer, 1x oligos trial from yesterday (yielded best PEG results)
- folding rxns:
32 (40 μL rxn) c5.0C (no latch, inside ligand)
32 (40 μL rxn) c5.0D (no latch, outside ligand)
8 (40 μL rxn) c5.0A (no latch, no ligand)
- repeat 8%, 10% PEG precipitation
Folding Reactions
- reconstituted the speedvaced 5.0.C and 5.0.D reactions from yesterday in 200 μL. Essentially we could have skipped the speedvac because as it turns out we're just going to be using the 30mM MgCl2. If we'd needed 20mM we would have reconstitued in 16 μL.
- Made more 10x folding buffer (300 mM MgCl2)
- folding rxns:
32 (40 μL rxn) c5.0C (no latch, inside ligand)
32 (40 μL rxn) c5.0D (no latch, outside ligand)
8 (40 μL rxn) c5.0A (no latch, no ligand)
- Each rxn:
16 ul oligos
9 ul p7308
11 ul H20
4 ul 10x folding buffer
PEG ppt
- 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 30 μL 1x folding buffer with 30 mM MgCl2
| lane | contents | loading dye |
| 1 | 8 μL untreated 5.0.A | 3 μL |
| 2 | 20 μL 5.0.A in 7% PEG and 0.5 M NaCl (supernatant) | 3 μL |
| 3 | 8 μL 5.0.A in 7% PEG and 0.5 M NaCl (pellet) | 3 μL |
| 4 | 20 μL 5.0.A in 8% PEG and 0.5 M NaCl (supernatant) | 3 μL |
| 5 | 8 μL 5.0.A in 8% PEG and 0.5 M NaCl (pellet) | 3 μL |
| 6 | 20 μL 5.0.A in 9% PEG and 0.5 M NaCl (supernatant) | 3 μL |
| 7 | 8 μL 5.0.A in 9% PEG and 0.5 M NaCl (pellet) | 3 μL |
| 8 | 20 μL 5.0.A in 10% PEG and 0.5 M NaCl (supernatant) | 3 μL |
| 9 | 8 μL 5.0.A in 10% PEG and 0.5 M NaCl (pellet) | 3 μL |
| 10 | 1 kb+ ladder | 3 μL |
| 11 | 2.7 μL p7308 | 3 μL |
| 12 | 8 μL untreated 5.0.C | 3 μL |
| 13 | 20 μL 5.0.C in 7% PEG and 0.5 M NaCl (supernatant) | 3 μL |
| 14 | 8 μL 5.0.C in 7% PEG and 0.5 M NaCl (pellet) | 3 μL |
| 15 | 20 μL 5.0.C in 8% PEG and 0.5 M NaCl (supernatant) | 3 μL |
| 16 | 8 μL 5.0.C in 8% PEG and 0.5 M NaCl (pellet) | 3 μL |
| 17 | 20 μL 5.0.C in 9% PEG and 0.5 M NaCl (supernatant) | 3 μL |
| 18 | 8 μL 5.0.C in 9% PEG and 0.5 M NaCl (pellet) | 3 μL |
| 19 | 20 μL 5.0.C in 10% PEG and 0.5 M NaCl (supernatant) | 3 μL |
| 20 | 8 μL 5.0.C in 10% PEG and 0.5 M NaCl (pellet) | 3 μL |
| 21 | 8 μL untreated 5.0.D | 3 μL |
| 22 | 20 μL 5.0.D in 7% PEG and 0.5 M NaCl (supernatant) | 3 μL |
| 23 | 8 μL 5.0.D in 7% PEG and 0.5 M NaCl (pellet) | 3 μL |
| 24 | 20 μL 5.0.D in 8% PEG and 0.5 M NaCl (supernatant) | 3 μL |
| 25 | 8 μL 5.0.D in 8% PEG and 0.5 M NaCl (pellet) | 3 μL |
| 26 | 20 μL 5.0.D in 9% PEG and 0.5 M NaCl (supernatant) | 3 μL |
| 27 | 8 μL 5.0.D in 9% PEG and 0.5 M NaCl (pellet) | 3 μL |
| 28 | 20 μL 5.0.D in 10% PEG and 0.5 M NaCl (supernatant) | 3 μL |
| 29 | 8 μL 5.0.D in 10% PEG and 0.5 M NaCl (pellet) | 3 μL |
| 30 | 1 kb+ ladder | 3 μL |
| 31 | 2.7 μL p7308 | 3 μL |
| 32-40 | (empty) | 3 μL |
