IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-7-20

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c4.0.b Folding

Stock oligos

  • Reconstituted biotinylated oligos (c4.0.4.1b-4b and c4.0.6.1b-4b) to 200uL.

Pre-working stocks

  • Mix 2.5 μL of each 200 μM stock oligo. 2.5μL biotinylated oligos + 7.5μL water go into each pre-working stock.
pre-working stock desc oligo tube labels total # of oligos
c4.0.4b barrel oligos @ outside aptamers -aptamers +biotin c4.0.4.1b-c4.0.4.4b 4
c4.0.6b barrel oligos @ inside aptamers -aptamers +biotin c4.0.6.1b-c4.0.6.4b 4

Make c4.0.b working stocks

  • NB: Due to miscommunication, the working stocks listed here (ie. 4.0Db, Eb, Fb, Gb) and those created the day after were folded with both core and latches together. They should have been separately folded.

Final concentration of each oligo in working stocks is 250 nM.

Stock ID Experiment 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 4b 6b
c4.0Db no latches, inside biotin x x x x x x x x
c4.0Eb no latches, outside biotin x x x x x x x x
c4.0Fb latch design 1, inside biotin x x x x x x x x
c4.0Gb latch design 1, outside biotin x x x x x x x x
  • NB: Due to running out of core pre-working stock 1, Hb and Ib were not created today, but will be tomorrow.

c4.0.b Folding Experiment

One reaction each with working stocks Db, Eb, Fb, Gb:

Reagents

  • 9 μL p7308 scaffold = (10 nM)/(44 nM) * 40 μL
  • 16 μL oligos = (100 nM)/(250 nM) * 40 μL
  • 4 μL 10x folding buffer (500 mM HEPES pH 7.5, 500 mM NaCl, 100 mM MgCl2)
  • 11 μL dH2O
  • total volume: 40 μL

Thermocycler

  • FOLDINGD program

Streptavidin-biotin binding experiments, continued

  • goal: show that streptavidin and biotin are binding and can be imaged with PAGE
    • show with both biotinylated oligos and biotinylated DNA nanostructures
lane streptavidin (2 μM) (μL) biotinylated oligo (1 μM) (μL) water (μL) loading buffer (10x Tris-glycine)
1 2 - 8 2
2 - 8 2 2
3 4 4 2 2
4 2 2 6 2
5 2 8 0 2
6 1 4 5 2
7-12 same as lanes 1-6
lane streptavidin (2 μM) (μL) box Ib (10 nM) (μL) water (μL) loading buffer (10x Tris-glycine)
1 2 - 8 2
2 - 4 6 2
3 2 2 6 2
4 1 1 8 2
5 1 4 5 2
6 0.5 2 7.5 2
7-12 same as lanes 1-6
12% PAGE, Coomassie imaging of oligo-streptavidin expt.
12% PAGE, DNA imaging of oligo-streptavidin expt.
12% PAGE, Coomassie imaging of nanostructure-streptavidin expt.
12% PAGE, DNA imaging of nanostructure-streptavidin expt.
  • results
    • results are inconclusive.
    • Coomassie imaging of oligo-streptavidin expt is faint
    • EtBr imaging of oligo-streptavidin expt is also faint, because ss DNA is difficult to image
    • Coomassie imaging of nanostructure-streptavidin expt is faint, and definitely shows no protein at the slowest band (which may be nanostructures)
    • EtBr imaging of nanostructure-streptavidin expt shows both nanostructures and oligos, but it is unclear which bands correspond to which