IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-16: Difference between revisions
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| 10||-ligand||10 {{ul}} 10 nM 8%-PEG-purified, ligand-incubated c5.0.A||2 {{ul}}||2 {{ul}}||1 {{ul}} 500 U/mL||5 {{ul}} | | 10||-ligand||10 {{ul}} 10 nM 8%-PEG-purified, ligand-incubated c5.0.A||2 {{ul}}||2 {{ul}}||1 {{ul}} 500 U/mL||5 {{ul}} | ||
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==Magnetic Streptavidin Protection, Take 2== | |||
* Steps 9-11 were done from the [[IGEM:Harvard/2006/DNA_nanostructures/Notebook/2006-8-15#Magnetic_Streptavidin_Protection.2C_Take_2|Tues 8.15 protocol]]. | |||
*'''Mistakes:''' | |||
# The Speedvac was allowed to proceed for an hour. Upon return, what had been thought to be 200uL of final-supernatant had completely dried - unless the tubes that had been speedvaced were not in fact the tubes that had contained the final-supernatant, but intermediate tubes. Because the initial final-supernatant had appeared slightly brown, these tubes were placed back into the MagnaRack and the supernatants were re-extracted into different, final tubes. These tubes might have accidentally been thrown out in the cleaning of the lab area that occured while I was out to lunch. If so, there might be some small residual amount of elute which might show up in the gels, but is unlikely to. If this is the case, the experiment will need to be repeated (cost of beads: ~$32). | |||
# If the tubes were the correct ones, the fact that they were Speedvaced to drying might have damaged the nanostructures irreparably and made them difficult, if not impossible to visualize in the gel. But hopefully scaffold and oligos will still be able to be seen. |
Revision as of 14:25, 16 August 2006
Repeat PEG Precipitation
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 |
Protection assay
- used nanostructures folded above that were purified with 8% PEG, 0.5 M NaCl
- mixed the following reactions, adding enzyme last
- attachment DNA used: c4.0.6.1ob (37 bp)
- oligo-ligand: script output (45 bp)
- incubated at 37[[:Category:{{{1}}}|{{{1}}}]] for 40 min.
- 2 μL loading dye added
- 20 μL each reaction run on 18% Tris-Gly PA gel for 1.5 h at 120 V, stained with SYBR Gold, imaged under EtBr filter
lane | trial | DNA | 10x NEBuffer 4 | 10x BSA | AscI | water |
1 | 10 bp+ ladder | 5 μL | ||||
2 | -nanostructures -ligand | 1 μL 1 μM attachment DNA | 2 μL | 2 μL | 1 μL 500 U/mL | 14 μL |
3 | -nanostructures -attachment | 1 μL 1 μM oligo-ligand | 2 μL | 2 μL | 1 μL 500 U/mL | 14 μL |
4 | -nanostructures -enzyme | 1 μL 1 μM attachment DNA, 1 μL 1 μM oligo-ligand | 2 μL | 2 μL | 0 μL | 14 μL |
5 | -nanostructures | 1 μL 1 μM attachment DNA, 1 μL 1 μM oligo-ligand | 2 μL | 2 μL | 1 μL 500 U/mL | 13 μL |
6 | outward-facing ligands | 10 μL 10 nM 8%-PEG-purified, ligand-incubated c5.0.C | 2 μL | 2 μL | 1 μL 500 U/mL | 5 μL |
7 | inward-facing ligands | 10 μL 10 nM 8%-PEG-purified, ligand-incubated c5.0.D | 2 μL | 2 μL | 1 μL 500 U/mL | 5 μL |
8 | -oligos | 2.25 μL 44 nM p7308 | 2 μL | 2 μL | 1 μL 500 U/mL | 13.75 μL |
9 | -enzyme | 10 μL 10 nM 8%-PEG-purified, ligand-incubated c5.0.C | 2 μL | 2 μL | 0 μL | 6 μL |
10 | -ligand | 10 μL 10 nM 8%-PEG-purified, ligand-incubated c5.0.A | 2 μL | 2 μL | 1 μL 500 U/mL | 5 μL |
Magnetic Streptavidin Protection, Take 2
- Steps 9-11 were done from the Tues 8.15 protocol.
- Mistakes:
- The Speedvac was allowed to proceed for an hour. Upon return, what had been thought to be 200uL of final-supernatant had completely dried - unless the tubes that had been speedvaced were not in fact the tubes that had contained the final-supernatant, but intermediate tubes. Because the initial final-supernatant had appeared slightly brown, these tubes were placed back into the MagnaRack and the supernatants were re-extracted into different, final tubes. These tubes might have accidentally been thrown out in the cleaning of the lab area that occured while I was out to lunch. If so, there might be some small residual amount of elute which might show up in the gels, but is unlikely to. If this is the case, the experiment will need to be repeated (cost of beads: ~$32).
- If the tubes were the correct ones, the fact that they were Speedvaced to drying might have damaged the nanostructures irreparably and made them difficult, if not impossible to visualize in the gel. But hopefully scaffold and oligos will still be able to be seen.