IGEM:Harvard/2006/DNA nanostructures/Notebook

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Contents

Daily notebook

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General Notes

  • Stock Oligos: reconstituted (50uM for non-biotinylated oligos, 200uM for biotinylated) oligos in wells or tubes
  • Pre-working Stocks: mixtures of 10uM of each oligo needed for a "class" of oligos (ex. core, barrel, lid1, lid2); for non-biot oligos, this requires 10uL of each of the necessary stock oligos, for biot-oligos, this requires 2.5uL of stock oligos + 7.5uL dH2O
  • Working Stocks: mixtures of however-many-oligos-are-in-each-class uL (ex. if there are 94 oligos in the core class of 3.2's design, use 94uL of the core pre-working stock), diluted to a total of 200uL of solution (each individual oligo will be at 250nM)

Brainstorming and future projects

Thrombin-aptamer experiments

Questions / procedures

  • what percent gel? 10% to 20% polyacrylamide gels, no SDS (but would make for a good control)
  • what incubation conditions?
  • how much protein and DNA? protein at 1 μM, DNA at 2 μM
  • Coomassie stain

Experiments

number thrombin aptamer nanotube DNA-stained prediction protein-stained prediction
0---no bandsno bands
1--+slow band (nanotube)no bands
2-+-fast band (aptamer)no bands
3-++slow band (aptamer-nanotube), traces of fast band (aptamer)no bands
4+--no bandsfast band (thrombin)
5+-+slow band (nanotube)fast band (thrombin)
6++-medium band (aptamer-thrombin), fast band (aptamer)medium band (aptamer-thrombin), traces of fast band (thrombin)
7+++very slow band (thrombin-aptamer-nanotube), slow band (aptamer-nantotube), traces of fast band (aptamer)very slow band (thrombin-aptamer-nanotube), medium band (aptamer-thrombin), traces of fast band (thrombin)

Buffers

  • Macaya's and Bock's selection buffer: 20 mM Tris-acetate, pH 7.4, 140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2
  • Liu's incubation buffer: 40 mM Tris, 20 mM CH3COOH, 2mM EDTA, 12.5 mM (CH3COO)2Mg, pH 8.0
  • Liu's PAGE buffer: 1x TAE/Mg2+
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