IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-15
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Mg2+, Oligo-Concentration Titration w/ c5.0 (cont'd)
3. PEG
PEG:
- GOAL: test 6%, 8%, 10% PEG precipitations
- incubated on ice for 15 min.
- spun at 16 k rcf at 4[[:Category:{{{1}}}|{{{1}}}]] for 10 min.
- carefully pipetted off supernatant
- resuspended "pellet" in 50 μL of water
-
Gel 1 after 45 minutes
-
Gel 2 after 45 minutes
-
Gel 1 after 75 minutes
-
Gel 2 after 75 minutes
Trial | Final PEG % | Lanes | 20% PEG (μL) | 5 M NaCl (μL) | Nanostructures (μL) | Water (μL) | Total volume (μL) |
1kb+ ladder | - | 1 | - | - | - | - | 10 |
p7308 | - | 2 | - | - | - | - | 10 |
1 | 0% | 3 (untreated trial 1) | - | - | - | - | - |
1-6 | 6% | 4 (pellet) | 15 | 5 | 20 | 10 | 50 |
1-6 | 6% | 5 (supernatant) | - | - | - | - | - |
1-8 | 8% | 6 (pellet) | 20 | 5 | 20 | 5 | 50 |
1-8 | 8% | 7 (supernatant) | - | - | - | - | - |
1-10 | 10% | 8 (pellet) | 25 | 5 | 20 | 0 | 50 |
1-10 | 10% | 9 (supernatant) | - | - | - | - | - |
2 | 0% | 10 (untreated trial 2) | - | - | - | - | - |
2-6 | 6% | 11 (pellet) | 15 | 5 | 20 | 10 | 50 |
2-6 | 6% | 12 (supernatant) | - | - | - | - | - |
2-8 | 8% | 13 (pellet) | 20 | 5 | 20 | 5 | 50 |
2-8 | 8% | 14 (supernatant) | - | - | - | - | - |
2-10 | 10% | 15 (pellet) | 25 | 5 | 20 | 0 | 50 |
2-10 | 10% | 16 (supernatant) | - | - | - | - | - |
3 | 0% | 17 (untreated trial 3) | - | - | - | - | - |
3-6 | 6% | 18 (pellet) | 15 | 5 | 20 | 10 | 50 |
3-6 | 6% | 19 (supernatant) | - | - | - | - | - |
1kb+ ladder | - | 1 | - | - | - | - | 10 |
p7308 | - | 2 | - | - | - | - | 10 |
3-8 | 8% | 3 (pellet) | 20 | 5 | 20 | 5 | 50 |
3-8 | 8% | 4 (supernatant) | - | - | - | - | - |
3-10 | 10% | 5 (pellet) | 25 | 5 | 20 | 0 | 50 |
3-10 | 10% | 6 (supernatant) | - | - | - | - | - |
4 | 0% | 7 (untreated trial 4) | - | - | - | - | - |
4-6 | 6% | 8 (pellet) | 15 | 5 | 20 | 10 | 50 |
4-6 | 6% | 9 (supernatant) | - | - | - | - | - |
4-8 | 8% | 10 (pellet) | 20 | 5 | 20 | 5 | 50 |
4-8 | 8% | 11 (supernatant) | - | - | - | - | - |
4-10 | 10% | 12 (pellet) | 25 | 5 | 20 | 0 | 50 |
4-10 | 10% | 13 (supernatant) | - | - | - | - | - |
5 | 0% | 14 (untreated trial 5) | - | - | - | - | - |
5-6 | 6% | 15 (pellet) | 15 | 5 | 20 | 10 | 50 |
5-6 | 6% | 16 (supernatant) | - | - | - | - | - |
5-8 | 8% | 17 (pellet) | 20 | 5 | 20 | 5 | 50 |
5-8 | 8% | 18 (supernatant) | - | - | - | - | - |
5-10 | 10% | 19 (pellet) | 25 | 5 | 20 | 0 | 50 |
5-10 | 10% | 20 (supernatant) | - | - | - | - | - |
1kb+ ladder | - | 1 | - | - | - | - | 10 |
p7308 | - | 2 | - | - | - | - | 10 |
6 | 0% | 3 (untreated trial 6) | - | - | - | - | |
6-6 | 6% | 4 (pellet) | 15 | 5 | 20 | 10 | 50 |
6-6 | 6% | 5 (supernatant) | - | - | - | - | - |
6-8 | 8% | 6 (pellet) | 20 | 5 | 20 | 5 | 50 |
6-8 | 8% | 7 (supernatant) | - | - | - | - | - |
6-10 | 10% | 8 (pellet) | 25 | 5 | 20 | 0 | 50 |
6-10 | 10% | 9 (supernatant) | - | - | - | - | - |
6hb-4 | 4% | 10 (pellet) | 10 | 5 | 20 | 15 | 50 |
6hb-4 | 4% | 11 (supernatant) | - | - | - | - | - |
6hb-6 | 6% | 12 (pellet) | 15 | 5 | 20 | 10 | 50 |
6hb-6 | 6% | 13 (supernatant) | - | - | - | - | - |
6hb-8 | 8% | 14 (pellet) | 20 | 5 | 20 | 5 | 50 |
6hb-8 | 8% | 15 (supernatant) | - | - | - | - | - |
6hb-10 | 10% | 16 (pellet) | 25 | 5 | 20 | 0 | 50 |
6hb-10 | 10% | 17 (supernatant) | - | - | - | - | - |
Revised protection assay protocol (proposed)
Folding
- use working stock that includes all latches, as well as oligo-ligand
- fold nanostructures with appropriate folding conditions
- appears to be either: 30 mM MgCl2 with 1x oligos or 20 mM MgCl2 with 6x, depending on PEG fractionation repeat experiment
Purification
- purify nanostructures with PEG precipitation using 7%, 8%, 9%, or 10%, depending on PEG fractionation repeat experiment
Digest
- total volume of each digest trial: 20 μL
trial | DNA | 10x NEBuffer 4 | 10x BSA | AscI | water |
experimental | 10 μL 10 nM purified, ligand-incubated nanostructures | 2 μL | 2μL | 1 μL 500 U/mL | 5 μL |
-oligos | 2.25 μL 44 nM p7308 (or appropriate scaffold) | 2 μL | 2 μL | 1 μL 500 U/mL | 13.75 μL |
-enzyme | 10 μL 10 nM purified, ligand-incubated nanostructures | 2 μL | 2 μL | 0 μL | 6 μL |
-ligand | 10 μL 10 nM purified nanostructures (no ligand) | 2 μL | 2 μL | 1 μL 500 U/mL | 5 μL |
-nanostructures -enzyme | 1 μL 1 μM attachment DNA, 1 μL 1 μM oligo-ligand | 2 μL | 2 μL | 0 μL | 14 μL |
-nanostructures | 1 μL 1 μM attachment DNA, 1 μL 1 μM oligo-ligand | 2 μL | 2 μL | 1 μL 500 U/mL | 13 μL |