IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-15: Difference between revisions
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** 5 {{ul}} water | ** 5 {{ul}} water | ||
** 1 {{ul}} 500 units/mL AscI | ** 1 {{ul}} 500 units/mL AscI | ||
* negative control: 2.25 {{ul}} p7308 (or other molar amount of scaffold) | * negative control: 2.25 {{ul}} p7308 (or other molar amount of scaffold), 2 {{ul}} 10x BSA, 2 {{ul}} 10x NEBuffer 4, 13.75 {{ul}} water | ||
* negative control: 10 {{ul}} 10 nM purified nanostructures | * negative control: 10 {{ul}} 10 nM purified nanostructures, 2 {{ul}} 10x BSA, 2 {{ul}} 10x NEBuffer 4, 6 {{ul}} water | ||
* negative control: 10 {{ul}} 10 nM purified nanostructures (with no oligo-ligand added), 2 {{ul}} 10x BSA, 2 {{ul}} 10x NEBuffer 4, 1 {{ul}} 500 units/mL AscI, 5 {{ul}} water | |||
* negative control: 1 {{ul}} 1 {{um}} ligand DNA, 1 {{ul}} 1 {{um}} attachment DNA, 2 {{ul}} 10x BSA, 2 {{ul}} 10x NEBuffer 4, 14 {{ul}} water | * negative control: 1 {{ul}} 1 {{um}} ligand DNA, 1 {{ul}} 1 {{um}} attachment DNA, 2 {{ul}} 10x BSA, 2 {{ul}} 10x NEBuffer 4, 14 {{ul}} water | ||
* positive control: 1 {{ul}} 1 {{um}} ligand DNA, 1 {{ul}} 1 {{um}} attachment DNA, 2 {{ul}} 10x BSA, 2 {{ul}} 10x NEBuffer 4, 1 {{ul}} 500 units/mL AscI, 13 {{ul}} water | * positive control: 1 {{ul}} 1 {{um}} ligand DNA, 1 {{ul}} 1 {{um}} attachment DNA, 2 {{ul}} 10x BSA, 2 {{ul}} 10x NEBuffer 4, 1 {{ul}} 500 units/mL AscI, 13 {{ul}} water | ||
* incubate all trials at 37{{c}} for 30 min. | * incubate all trials at 37{{c}} for 30 min. | ||
Revision as of 12:32, 15 August 2006
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 nanostructure digest: 20 μL
- 100 fmols (~450 ng) purified, ligand-incubated nanostructures (MW ~= 4,500,000 Da) = 10 μL 10 nM
- 2 μL 10x BSA
- 2 μL 10x NEBuffer 4
- 5 μL water
- 1 μL 500 units/mL AscI
- negative control: 2.25 μL p7308 (or other molar amount of scaffold), 2 μL 10x BSA, 2 μL 10x NEBuffer 4, 13.75 μL water
- negative control: 10 μL 10 nM purified nanostructures, 2 μL 10x BSA, 2 μL 10x NEBuffer 4, 6 μL water
- negative control: 10 μL 10 nM purified nanostructures (with no oligo-ligand added), 2 μL 10x BSA, 2 μL 10x NEBuffer 4, 1 μL 500 units/mL AscI, 5 μL water
- negative control: 1 μL 1 μM ligand DNA, 1 μL 1 μM attachment DNA, 2 μL 10x BSA, 2 μL 10x NEBuffer 4, 14 μL water
- positive control: 1 μL 1 μM ligand DNA, 1 μL 1 μM attachment DNA, 2 μL 10x BSA, 2 μL 10x NEBuffer 4, 1 μL 500 units/mL AscI, 13 μL water
- incubate all trials at 37[[:Category:{{{1}}}|{{{1}}}]] for 30 min.
