IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-7-25

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Oligos Order Form

c5.0 Partial Oligo Order Form


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
20060725 peg.jpg
  • 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
The streptavidin band appears at half the motility of the dye band (which was bleached to white after silver staining).
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)