IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-11: 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}}) | ||
|} | |} | ||
==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. | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Trial''' | |||
| align="center" style="background:#f0f0f0;"|'''Final PEG %''' | |||
| align="center" style="background:#f0f0f0;"|'''20% PEG ({{ul}})''' | |||
| align="center" style="background:#f0f0f0;"|'''5 M NaCl ({{ul}})''' | |||
| align="center" style="background:#f0f0f0;"|'''Nanostructures ({{ul}})''' | |||
| align="center" style="background:#f0f0f0;"|'''water ({{ul}})''' | |||
| align="center" style="background:#f0f0f0;"|'''Total volume ({{ul}})''' | |||
|- | |||
| 1-0||0%||0||5||5||20||50 | |||
|- | |||
| 1-2||2%||5||5||5||15||50 | |||
|- | |||
| 1-4||4%||10||5||5||10||50 | |||
|- | |||
| 1-6||6%||15||5||5||5||50 | |||
|- | |||
| 1-8||8%||20||5||5||0||50 | |||
|- | |||
| 2-0||0%||0||5||5||20||50 | |||
|- | |||
| 2-2||2%||5||5||5||15||50 | |||
|- | |||
| 2-4||4%||10||5||5||10||50 | |||
|- | |||
| 2-6||6%||15||5||5||5||50 | |||
|- | |||
| 2-8||8%||20||5||5||0||50 | |||
|- | |||
| 3-0||0%||0||5||5||20||50 | |||
|- | |||
| 3-2||2%||5||5||5||15||50 | |||
|- | |||
| 3-4||4%||10||5||5||10||50 | |||
|- | |||
| 3-6||6%||15||5||5||5||50 | |||
|- | |||
| 3-8||8%||20||5||5||0||50 | |||
|- | |||
| 4-0||0%||0||5||5||20||50 | |||
|- | |||
| 4-2||2%||5||5||5||15||50 | |||
|- | |||
| 4-4||4%||10||5||5||10||50 | |||
|- | |||
| 4-6||6%||15||5||5||5||50 | |||
|- | |||
| 4-8||8%||20||5||5||0||50 | |||
|- | |||
| 5-0||0%||0||5||5||20||50 | |||
|- | |||
| 5-2||2%||5||5||5||15||50 | |||
|- | |||
| 5-4||4%||10||5||5||10||50 | |||
|- | |||
| 5-6||6%||15||5||5||5||50 | |||
|- | |||
| 5-8||8%||20||5||5||0||50 | |||
|- | |||
| 6-0||0%||0||5||5||20||50 | |||
|- | |||
| 6-2||2%||5||5||5||15||50 | |||
|- | |||
| 6-4||4%||10||5||5||10||50 | |||
|- | |||
| 6-6||6%||15||5||5||5||50 | |||
|- | |||
| 6-8||8%||20||5||5||0||50 | |||
|} | |||
* incubate on ice for 15 min. | |||
* spin at 16 k rcf at 4{{c}} for 10 min. | |||
* carefully pipet off supernatant | |||
* resuspend "pellet" in 10 {{ul}} of respective folding buffer |
Revision as of 11:18, 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).
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 % | 20% PEG (μL) | 5 M NaCl (μL) | Nanostructures (μL) | water (μL) | Total volume (μL) |
1-0 | 0% | 0 | 5 | 5 | 20 | 50 |
1-2 | 2% | 5 | 5 | 5 | 15 | 50 |
1-4 | 4% | 10 | 5 | 5 | 10 | 50 |
1-6 | 6% | 15 | 5 | 5 | 5 | 50 |
1-8 | 8% | 20 | 5 | 5 | 0 | 50 |
2-0 | 0% | 0 | 5 | 5 | 20 | 50 |
2-2 | 2% | 5 | 5 | 5 | 15 | 50 |
2-4 | 4% | 10 | 5 | 5 | 10 | 50 |
2-6 | 6% | 15 | 5 | 5 | 5 | 50 |
2-8 | 8% | 20 | 5 | 5 | 0 | 50 |
3-0 | 0% | 0 | 5 | 5 | 20 | 50 |
3-2 | 2% | 5 | 5 | 5 | 15 | 50 |
3-4 | 4% | 10 | 5 | 5 | 10 | 50 |
3-6 | 6% | 15 | 5 | 5 | 5 | 50 |
3-8 | 8% | 20 | 5 | 5 | 0 | 50 |
4-0 | 0% | 0 | 5 | 5 | 20 | 50 |
4-2 | 2% | 5 | 5 | 5 | 15 | 50 |
4-4 | 4% | 10 | 5 | 5 | 10 | 50 |
4-6 | 6% | 15 | 5 | 5 | 5 | 50 |
4-8 | 8% | 20 | 5 | 5 | 0 | 50 |
5-0 | 0% | 0 | 5 | 5 | 20 | 50 |
5-2 | 2% | 5 | 5 | 5 | 15 | 50 |
5-4 | 4% | 10 | 5 | 5 | 10 | 50 |
5-6 | 6% | 15 | 5 | 5 | 5 | 50 |
5-8 | 8% | 20 | 5 | 5 | 0 | 50 |
6-0 | 0% | 0 | 5 | 5 | 20 | 50 |
6-2 | 2% | 5 | 5 | 5 | 15 | 50 |
6-4 | 4% | 10 | 5 | 5 | 10 | 50 |
6-6 | 6% | 15 | 5 | 5 | 5 | 50 |
6-8 | 8% | 20 | 5 | 5 | 0 | 50 |
- 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 10 μL of respective folding buffer