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

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Contents

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
  • 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).
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)
110 μL 4 μM40,00002
210 μL 2 μM20,00002
35 μL 2 μM10,00052
45 μL 1 μM5,00052
55 μL 0.5 μM2,50052
62 μL 0.5 μM1,00082
71 μL 0.5 μM50092
82.5 μL 0.1 μM2507.52
91 μL 0.1 μM10092
101 μL 0.05 μM5092
  • 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)
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