IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-11: Difference between revisions
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Protocol: prepare the following 30 samples. | Protocol: prepare the following 30 samples. | ||
[[Image:2006-08- | [[Image:2006-08-11_18hr_03min.jpg|thumb|Gel 1]] | ||
[[Image:2006-08- | [[Image:2006-08-11_18hr_05min.jpg|thumb|Gel 2]] | ||
{| {{table}} | {| {{table}} | ||
Revision as of 15:03, 11 August 2006
Thoughts/ramblings/goals/questions/general frustrations
The results of yesterday's experiment show that Microcon filtration gives low yields and the PEG precipitation (at least at 10%) damages nanostructures regardless of folding conditions.
Questions
- Are Microcon yields unacceptably low, or are they acceptable? (Can we use the NanoDrop to quantify our yield?)
- Gels are much better for quantifying yield - you can try both and see how they compare.
- Low concentrations of PEG should precipitate large nanostructures. Will some smaller concentration of PEG not harm nanostructures formed under some folding conditions?
- Our August 2 experiment showed that even low concentrations of PEG damage nanostructures folded under "standard" conditions (10x oligos, 10 mM MgCl2).
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).
0.5x TBE, 11 mM MgCl2
| 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) |
Gigundo PEG precipitation
- goal: test 0% to 8% PEG precipitations with nanostructures folded under all six folding conditions from yesterday
- optimistic hypothesis: nanostructures folded with higher concentrations of oligos and/or MgCl2 will show less damage after treatment with PEG
Protocol: prepare the following 30 samples.
| Trial | Final PEG % | Gel | Lanes | 20% PEG (μL) | 5 M NaCl (μL) | Nanostructures (μL) | water (μL) | Total volume (μL) |
| 1-0 | 0% | 1 | 1 | 0 | 5 | 5 | 20 | 50 |
| 1-2 | 2% | 1 | 3 | 5 | 5 | 5 | 15 | 50 |
| 1-4 | 4% | 1 | 5 | 10 | 5 | 5 | 10 | 50 |
| 1-6 | 6% | 1 | 7 | 15 | 5 | 5 | 5 | 50 |
| 1 kb+ ladder | 1 | 9 | 10 | |||||
| p7308 | 1 | 10 | 10 | |||||
| 1-8 | 8% | 1 | 11 | 20 | 5 | 5 | 0 | 50 |
| 2-0 | 0% | 1 | 13 | 0 | 5 | 5 | 20 | 50 |
| 2-2 | 2% | 1 | 15 | 5 | 5 | 5 | 15 | 50 |
| 2-4 | 4% | 1 | 17 | 10 | 5 | 5 | 10 | 50 |
| 2-6 | 6% | 1 | 19 | 15 | 5 | 5 | 5 | 50 |
| 2-8 | 8% | 1 | 21 | 20 | 5 | 5 | 0 | 50 |
| 3-0 | 0% | 1 | 23 | 0 | 5 | 5 | 20 | 50 |
| 3-2 | 2% | 1 | 25 | 5 | 5 | 5 | 15 | 50 |
| 3-4 | 4% | 1 | 27 | 10 | 5 | 5 | 10 | 50 |
| 1 kb+ ladder | 1 | 29 | 10 | |||||
| p7308 | 1 | 30 | 10 | |||||
| 3-6 | 6% | 1 | 31 | 15 | 5 | 5 | 5 | 50 |
| 3-8 | 8% | 1 | 33 | 20 | 5 | 5 | 0 | 50 |
| 4-0 | 0% | 1 | 35 | 0 | 5 | 5 | 20 | 50 |
| 4-2 | 2% | 1 | 37 | 5 | 5 | 5 | 15 | 50 |
| 4-4 | 4% | 1 | 39 | 10 | 5 | 5 | 10 | 50 |
| 4-6 | 6% | 2 | 1 | 15 | 5 | 5 | 5 | 50 |
| 4-8 | 8% | 2 | 3 | 20 | 5 | 5 | 0 | 50 |
| 5-0 | 0% | 2 | 5 | 0 | 5 | 5 | 20 | 50 |
| 5-2 | 2% | 2 | 7 | 5 | 5 | 5 | 15 | 50 |
| 1 kb+ ladder | 1 | 29 | 10 | |||||
| p7308 | 1 | 30 | 10 | |||||
| 5-4 | 4% | 2 | 11 | 10 | 5 | 5 | 10 | 50 |
| 5-6 | 6% | 2 | 13 | 15 | 5 | 5 | 5 | 50 |
| 5-8 | 8% | 2 | 15 | 20 | 5 | 5 | 0 | 50 |
| 6-0 | 0% | 2 | 17 | 0 | 5 | 5 | 20 | 50 |
| 6-2 | 2% | 2 | 19 | 5 | 5 | 5 | 15 | 50 |
| 6-4 | 4% | 2 | 21 | 10 | 5 | 5 | 10 | 50 |
| 6-6 | 6% | 2 | 23 | 15 | 5 | 5 | 5 | 50 |
| 6-8 | 8% | 2 | 25 | 20 | 5 | 5 | 0 | 50 |
| 1 kb+ ladder | 1 | 29 | 10 | |||||
| p7308 | 1 | 30 | 10 |
- incubate on ice for 15 min.
- spin at 16 k rcf at 4[[:Category:{{{1}}}|{{{1}}}]] for 10 min.
- carefully pipet off supernatant
- resuspend "pellet" in 20 μL of respective folding buffer
- load resuspended pellets in odd-numbered lanes of 2% TBE agarose gel supplemented to 10 mM MgCl2
- load 30 μL (of 50 μL) of supernatant into adjacent even-numbered lanes (e.g., trial 1-0 has pellet in lane 1 and supernatant in lane 2)
- run at 60V for 1 h
Results/discussion
- PEG precipitations appeared to have failed: no oligos were separated
- curiously, the dye in the "supernatant" lanes ran at about 2/3 of the speed of the dye in the "pellet" lanes, and it gave a smear and not a band
