IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-5

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Superconcentration Experiments

PAGE Coomassie Gel Imaging of Superconcentration of Eb, Fb, and Gb

  • destained: 10 min in H2O, two times
  • imaged gel from yesterday
lane component concentration amount of component in well μL streptavidin added μL
1 streptavidin 2uM 3.5 -
2 p7308 44uM 7.5 3.5
3 c4.0.6b (ie. biotinylated inside-oligos) 50nM each oligo (as in all pre-working stocks) 7 3.5
4 Eb PEG-pellet 21/8 of a rxn 15 3.5
5 Fb PEG-pellet 21/8 of a rxn 15 3.5
6 Gb PEG-pellet 21/8 of a rxn 15 3.5
7 streptavidin 2uM 7 -
12% PAGE run at 100V for 1hr10m coomassie-stained overnight
  • Conclusions:
    • streptavidin band clear
    • biotinylated oligos coomassie band clear too, in different place
      • there are definitely streptavidin-bound biotinylated oligos in lane 3
    • it is difficult to see in the image, but a darker blue band was visible at the top of lane 5, where the nanostructure should be
      • streptavidin bound to Eb
      • unfortunately, Eb = inside-biotinylation -latches, so how the streptavidin bound and was visualized when the Gb (outside-biotinylation +latch1) didn't bind it is questionable
      • it is a very faint band
    • no bands were seen at the top of the gel in the expected lane 6 (Gb)
  • is it possible that streptavidin actually bound to nanostructures can't be stained w/ coomassie?
    • that would explain why the inside-biotinylated +latch1 design, which would not (we expect) permit the biotinylated-oligos within to bind streptavidin, shows a lot more unbound streptavidin and biotinylated-oligos-bound streptavidin


PAGE EtBr Gel Imaging of Superconcentration of Eb, Fb, and Gb

  • as a first try, the gel was destained a further 30min in H2O before EtBr staining was attempted
  • 200mL of H2O +7uL of EtBr were used as a stain; the gel was allowed to stain for 15 min
  • no luck, no bands at all were seen under UV
    • will save gel in H2O till figure out if there is anything to be done about it

More Folding Reactions for Further Superconcentration Experiments with Stains-All

  • made working stocks (=12.5 rxns) for:
    • outside-biotinylation w/o latches, Eb
    • outside-biotinylation w/ "future" (aka four-oligo, aka latch2, aka staple latches) latches, Ib
      • latching which can be attempted post-incubation
      • can be incubated at 37°C for 1hr with the zipper oligos (c4.0.23, 24, 25), though latches can be used in the first incubation
    • inside-biotinylation w/o latches, Db
    • inside-biotinylation w/ latches, Hb
Stock ID Experiment 1 2 3 4 4b 5 6 6b 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 total H2O needed to 200uL
c4.0Eb outside biotin -latches 66uL 33 33 4 4 4 2 2 148 52
c4.0Ib outside biotin +latch design 2 (ie. 4-oligo) 66uL 33 33 4 4 4 2 2 148 52
c4.0Db inside biotin -latches 66uL 33 33 4 4 4 2 2 148 52
c4.0Hb inside biotin +latch design 2 (ie. 4-oligo) 66uL 33 33 4 4 4 2 2 148 52
  • folded according to given protocol, using FOLDINGD

Folding for MgCl2 and 6x Titrations, Take Two

  • because the gel from yesterday of the previoius titration showed no bands whatsoever, the reactions were refolded as before
  • the reactions were, as usual, folded with the FOLDINGD program

3D models continued

Added scales to two images; Sketchup file now posted under Design 5 schematics.