IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-7-23

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Nanostructure-streptavidin binding experiments

Because purification/precipitation experiments have been unsuccessful, and I don't want to waste any more time, I increased the amount of streptaviding we'd been planning to run by 10x and ran it anyway.Matthewmeisel 19:57, 23 July 2006 (EDT)

  • goals:
    • run purified DNA nanostructures on PA gel to determine its gel motility
    • run scaffold and streptavidin together on PA gel to show that there is no scaffold-streptavidin interaction (we hope!)
    • run concentrated, purified DNA nanostructures on a PA gel, with and without streptavidin, with biotin sites facing inwards and outwards, with and without lid latches that are added before streptavidin incubation
    • lanes to be run: (total volume of each: 17 μL)

Pre-working stocks

lane streptavidin (2 μM) (μL) p7308 scaffold
(44 nM)
(μL)
oligos (100 nM) (μL) nanostructures
(~10 nM in nanostructures,
~90 nM in unused oligos)
(μL)
water (μL) 1x DNA ladder in 1x TBE/glycerol loading dye (μL) 10x Tris-gly loading dye (μL)
1----710-
22.5---12.5-2
3-11.4--3.6-2
42.511.4--1.1-2
5--5.0 (3.2.7.2b)-10-2
62.5-5.0 (3.2.7.2b)-7.5-2
7---10.0 4.0.Eb5-2
82.5--10.0 4.0.Eb2.5-2
92.5--10.0 4.0.Ib2.5-2
10---10.0 4.0.Db5-2
112.5--10.0 4.0.Db2.5-2
122.5--10.0 4.0.Hb2.5-2
  • original plan:
    1. ladder
    2. streptavidin (2.5 μL 2.0 μM)
    3. scaffold (11.4 μL 44 nM)
    4. streptavidin (1.0 μL 0.5 μM) + scaffold (11.4 μL 44 nM)
    5. biotinylated oligos (5 μL 100 nM)
    6. biotinylated oligos (5 μL 100 nM) + streptavidin (1.0 μL 0.5 μM)
    7. nanostructure with outward-facing biotinylated oligos (10 μL 10 nM 4.0.Eb)
    8. nanostructure with outward-facing biotinylated oligos (10 μL 10 nM 4.0.Eb) + streptavidin (1.0 μL 0.5 μM)
    9. nanostructure with outward-facing biotinylated oligos (10 μL 10 nM 4.0.Ib) and latch + streptavidin (1.0 μL 0.5 μM)
    10. nanostructure with inward-facing biotinylated oligos (10 μL 10 nM 4.0.Db)
    11. nanostructure with inward-facing biotinylated oligos (10 μL 10 nM 4.0.Db) + streptavidin (1.0 μL 0.5 μM)
    12. nanostructure with inward-facing biotinylated oligos (10 μL 10 nM 4.0.Hb) and latch + streptavidin (1.0 μL 0.5 μM) (latch added before streptavidin incubation)
12% PA gel electrophoresis, stained with EtBr. The DNA ladder overflowed its well and into lanes 2-4.
12% PA gel electrophoresis, stained with EtBr. The DNA ladder overflowed its well and into lanes 2-4.
12% PA gel electrophoresis, stained with Coomassie blue.
12% PA gel electrophoresis, stained with Coomassie blue.
  • run on 12% PA gel, 120V at 4[[:Category:{{{1}}}|{{{1}}}]] for 50 min.
    • checks at 30 min. and at 50 min. shows that there is no visible dye migration between 30 min. and 50 min.
    • stained with 5 μL EtBr in 100 mL water
  • assumptions:
    • the incubation conditions required for successful streptavidin-biotin are essentially that the two species need to be mixed at room temperature and that the required incubation time is no longer than a few minutes
  • results:
    • streptavidin-bound DNA nanostructures show no gel shift (lanes 7-12)
    • no streptavidin is visible where DNA nanostructures are located at the top of the gel (lanes 8-9, 11-12)
      • probably because streptavidin concentration is too small
    • streptavidin-bound oligos shift upward (lanes 8-9, 11-12)
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