IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-7-19

Incubation and dilution protocol, take 2

 * Goal: to see the lower threshold of imaging streptavidin bound to biotin
 * and to see if we can image any streptavidin-biotin construct on a gel at all
 * Protocol: mix 6 2  streptavidin with 6  1  c.3.2.7.2b oligo, to give a final oligo concentration of 500 nM, and incubate at room temperature for 5 min.
 * dilution and electrophoresis:
 * each lane contained 2 of different dilutions (as per table below), 6  of water, and 2  of loading dye
 * lanes 7-12 were loaded with the same mixtures as lanes 1-6, respectively
 * ran on native polyacrylamide gel for 30 min. at 120V
 * no DNA imaged with EtBr staining
 * GelCode Blue staining shows a band in lane 3 with the same motility as control streptavidin in lane 1
 * this is probably free streptavidin
 * unclear where streptavidin-biotin complex is
 * expt appears to be consistent with manufacturer's notes that the lower detection limit is 8 ng (151 fmols streptavidin, MW=52.8 kDa)

Imaging biotinylated oligos

 * goal: show that we can image biotinylated oligos on a PA gel
 * run on a 12% native PA gel at 120V for 15 min.
 * clear bands appear in lanes 1-4
 * faint bands in lanes 5-6 are likely artifacts, as lanes 7-12 (empty) contain them as well

Brainstorming for future assay
Run two experiments in parallel, the first a control.
 * 1) assmebly
 * 2) * assemble a nanostructure with outward- (1) and inward- (2) facing biotinylated oligos
 * 3) * incubate assembled nanostructures with fluorescently-labeled streptavidin
 * 4) * close the container lids
 * 5) baseline protein assay
 * 6) * precipitate nanostructures
 * 7) * pour off supernatant (containing excess streptavidin)
 * 8) * resuspend nanostructures in water
 * 9) * measure streptavidin concentrations in each container (pre-digest)
 * 10) ** these are baseline values, and we expect that they should be similar if incubation efficencies for each nanostructure are similar
 * 11) protease digest
 * 12) * digest each nanostructure with protease
 * 13) ** particular protease and protocol must be finalized
 * 14) measure streptavidin concentrations in each container (post-digest)
 * 15) * ''these values should differ if biotinylated streptavidin can still be digested (container 1) and if a closed nanostructure protects its cargo (container 2)
 * 16) confirmation of presence of streptavidin
 * 17) * treat both containers with DNAse
 * 18) * measure streptavidin concentrations
 * 19) ** streptavidin is expected to be bound to biotin, but biotin not bound to oligos
 * 20) * treat both containers with protease
 * 21) * measure streptavidin concentrations
 * 22) ** values should all be close to zero

Container 4.0
Reagents (each expt in respective 0.2 mL PCR tube) Annealing protocol
 * start at 80
 * 60 cycles: wait 2 minutes, decrease 1
 * hold at 4

Gel analysis

 * 2% agarose gel supplemented to 10 mM and with 3  10 mg/mL EtBr (100 mL gel)
 * run in 1x TBE supplemented to 10 mM
 * 45 min at 130 V
 * results