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

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	-	-	-	-	7	10	-
2	2.5	-	-	-	12.5	-	2
3	-	11.4	-	-	3.6	-	2
4	2.5	11.4	-	-	1.1	-	2
5	-	-	5.0 (3.2.7.2b)	-	10	-	2
6	2.5	-	5.0 (3.2.7.2b)	-	7.5	-	2
7	-	-	-	10.0 4.0.Eb	5	-	2
8	2.5	-	-	10.0 4.0.Eb	2.5	-	2
9	2.5	-	-	10.0 4.0.Ib	2.5	-	2
10	-	-	-	10.0 4.0.Db	5	-	2
11	2.5	-	-	10.0 4.0.Db	2.5	-	2
12	2.5	-	-	10.0 4.0.Hb	2.5	-	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 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)