IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-15: Difference between revisions
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| 8||p7308 060522, 1:2 dil (12 {{ul}})||AGLB (2 {{ul}}) | | 8||p7308 060522, 1:2 dil (12 {{ul}})||AGLB (2 {{ul}}) | ||
|} | |} | ||
==Magnetic Streptavidin Bead "Protection" Assay Gel== | |||
*'''Gel''': | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Lane''' | |||
| align="center" style="background:#f0f0f0;"|'''Component''' | |||
| align="center" style="background:#f0f0f0;"|'''Amount''' | |||
|- | |||
| 1||water control||20uL | |||
|- | |||
| 2||c5.0.8(b)||20uL | |||
|- | |||
| 3||c5.0.E(b)||20uL | |||
|- | |||
| 4||c5.0.F(b)||20uL | |||
|- | |||
| 5||1kb+ ladder|| | |||
|- | |||
| 6||p7308|| | |||
|- | |||
| | |||
|} | |||
<gallery> | |||
Image:TC-20060815-strept-beads.tif| <br>2% agarose, EtBr, 10mM MgCl2 | |||
</gallery> |
Revision as of 14:00, 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
- note: we added PEG first, nanostructures last; mixed using tapping after everything was added; let it sit for like...5 min. before putting it on ice; let it sit a bit (ie. 10 min.) after centrifuging
-
Gel 1 after 45 minutes
-
Gel 2 after 45 minutes
-
Gel 1 after 75 minutes
-
Gel 2 after 75 minutes
RESULTS:
- decent separation of pellet and oligos for most of the trials
- looking at gels after 75 min. seems to show that the nanostructures are going into the gel, so we don't think they're getting denatured
- best trials seem to be: Trial 4 (20 mM MgCl2 w/ 6x oligos) and Trial 5 (30 mM MgCl2 w/ 1x oligos), each at 8% and 10% PEG
- next step: we will repeat this experiment in larger quantities to prepare for a protection assay
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 | 2 μL | 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 |
Quantitation
p7308 quantitation
- Speedvac 060522 p7308 sample down to 50% volume. This should remove any ethanol, and give you a slightly more manageable volume.
- Pour 2% agraose, 11 mM MgCl2 gel
- For gel loading make 1:2 dilution (add 20 μL of p7308 to 20 μL dH2O). Original estimate for 060522 prep was 42 nM, so hopefully it should correspond pretty well to 44 nM sample.
- Load gel according to table below
- Run for 2 hrs, 70V
- When imaging gel, use spot density tool to measure intensity of each band
- Use saturation indicator to take a picture just below the point where any bands start saturating on the image
- Draw a rectangle that fits around the largest band on the gel
- Copy that rectangle and position it directly above the first band. This will be used to measure background
- Repeat this for every band on the gel (one box for the band, one box for background)
- Record this data along with gel picture on the wiki
- To determine p7308 concentration, use background-subtracted value for each volume. Scale each unknown concentration against the control (44 nM) according to the ratio of background-subtracted intensity for the band that looks closest in intensity
- For example, if the band in lane 1 (3 μL, 44 nM) had an intensity of 1000, and the band in lane 5 (3 μL, ?? nM) had an intensity of 900, then we would record 900/1000 * 44 = 39.6 nM as the estimated concentration for that lane. Repeating for each lane should give you 3 data points, which you can average (throwing out any obvious outliers).
Lane | Contents | Loading Buffer |
0 | 1kb DNA ladder (5 μL) | |
1 | p7308 060323, 44 nM (3 μL) | AGLB (2 μL) + dH2O (9 μL) |
2 | p7308 060323, 44 nM (6 μL) | AGLB (2 μL) + dH2O (6 μL) |
3 | p7308 060323, 44 nM (9 μL) | AGLB (2 μL) + dH2O (3 μL) |
4 | p7308 060522, 1:2 dil (1 μL) | AGLB (2 μL) + dH2O (11 μL) |
5 | p7308 060522, 1:2 dil (3 μL) | AGLB (2 μL) + dH2O (9 μL) |
6 | p7308 060522, 1:2 dil (6 μL) | AGLB (2 μL) + dH2O (6 μL) |
7 | p7308 060522, 1:2 dil (9 μL) | AGLB (2 μL) + dH2O (3 μL) |
8 | p7308 060522, 1:2 dil (12 μL) | AGLB (2 μL) |
Magnetic Streptavidin Bead "Protection" Assay Gel
- Gel:
Lane | Component | Amount |
1 | water control | 20uL |
2 | c5.0.8(b) | 20uL |
3 | c5.0.E(b) | 20uL |
4 | c5.0.F(b) | 20uL |
5 | 1kb+ ladder | |
6 | p7308 | |
-
2% agarose, EtBr, 10mM MgCl2