IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-7-20: Difference between revisions

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| 7-12||colspan="4"|same as lanes 1-6
| 7-12||colspan="4"|same as lanes 1-6
|}
|}
[[Image:20060720_oligos-protein.jpg|thumb|12% PAGE, Coomassie imaging of oligo-streptavidin expt.]]
[[Image:20060720_oligo-dna.jpg|thumb|12% PAGE, DNA imaging of oligo-streptavidin expt.]]
[[Image:20060720_nano-protein.jpg|thumb|12% PAGE, Coomassie imaging of nanostructure-streptavidin expt.]]
[[Image:20060720_nano-dna.jpg|thumb|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

Revision as of 14:24, 20 July 2006

c4.0.b Folding

Stock oligos

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

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

Working Stocks for Biotinylated Oligos

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
c4.0Hb latch design 2, inside biotin x x x x x x x x x x x
c4.0Ib latch design 2, outside biotin x x x x x x x x x x x

c4.0.b Folding Experiment

Reagents

  • 9 μL p7308 scaffold = (10 nM)/(44 nM) * 40 μL
  • 16 μL oligos (3.2.A) = (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


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
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