IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-7-25
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Purification
PEG precipitation
- Used samples of 3.2.H and 3.2.I folded yesterday
- 20 μL of each, 190 mL water, 50 mL 20% PEG / 2.5 M NaCl in respective 500 μL PCR tubes
- gel analysis (2% agarose):
- lanes 3 and 6 (reconstituted precipitate of H and I, respectively) appear to be free of oligos (compare to lanes 2 and 5, which are raw folding reaction), but yields are still very low, as evidenced by amount of nanostructures in supernatant (lanes 4 and 7)
Gel purification
- 20 mL each of 3.2.D and 3.2.E run on a 2% agarose gel
- nanostructure bands isolated and purified using Qiagen gel purification kit (good for fragments up to 10 kb according to manufacturer)
- will run on agarose gel tomorrow to determine purity
Silver staining experiments
- goal: to determine the minimum concentration of streptavidin that can be visualized with silver stain, and to note the times at which different concentrations become visible
- methods: 12% PA gel with lanes listed below, run for 20 min. at 130 V, then stained with Bio-Rad Silver Stain Plus Kit and protocol.
- 35 min. in fixative solution
- 2 x 10 min. wash in water
- XX min. in staining solution
- 15 min. in stopping solution
lane | streptavidin | water (μL) | 10x Tris-gly loading dye (μL) | |
(μL) | (fmols) | |||
1 | 10 μL 4 μM | 40,000 | 0 | 2 |
2 | 10 μL 2 μM | 20,000 | 0 | 2 |
3 | 5 μL 2 μM | 10,000 | 5 | 2 |
4 | 5 μL 1 μM | 5,000 | 5 | 2 |
5 | 5 μL 0.5 μM | 2,500 | 5 | 2 |
6 | 2 μL 0.5 μM | 1,000 | 8 | 2 |
7 | 1 μL 0.5 μM | 500 | 9 | 2 |
8 | 2.5 μL 0.1 μM | 250 | 7.5 | 2 |
9 | 1 μL 0.1 μM | 100 | 9 | 2 |
10 | 1 μL 0.05 μM | 50 | 9 | 2 |
- results:
- first three lanes (40k, 20k, 10k pmol) stain reliably
- fourth and fifth lane lane (5k, 2.5k pmol) barely stains
- the rest of the lanes (1.25k pmol and less) do not stain more than the background
- this is disappointing (we can image 1 pmol of streptavidin with Coomassie blue)