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 )

Pre-working stocks

 * original plan:
 * ladder
 * streptavidin (2.5 2.0 )
 * scaffold (11.4 44 nM)
 * streptavidin (1.0 0.5 ) + scaffold (11.4  44 nM)
 * biotinylated oligos (5 100 nM)
 * biotinylated oligos (5 100 nM) + streptavidin (1.0  0.5 )
 * nanostructure with outward-facing biotinylated oligos (10 10 nM 4.0.Eb)
 * nanostructure with outward-facing biotinylated oligos (10 10 nM 4.0.Eb) + streptavidin (1.0  0.5 )
 * nanostructure with outward-facing biotinylated oligos (10 10 nM 4.0.Ib) and latch + streptavidin (1.0  0.5 )
 * nanostructure with inward-facing biotinylated oligos (10 10 nM 4.0.Db)
 * nanostructure with inward-facing biotinylated oligos (10 10 nM 4.0.Db) + streptavidin (1.0  0.5 )
 * nanostructure with inward-facing biotinylated oligos (10 10 nM 4.0.Hb) and latch + streptavidin (1.0  0.5 ) (latch added before streptavidin incubation)
 * run on 12% PA gel, 120V at 4 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 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)