IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-7-19: Difference between revisions
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* run on a 12% native PA gel at 120V for 15 min. | * run on a 12% native PA gel at 120V for 15 min. | ||
====Brainstorming for future assay==== | |||
Run two experiments in parallel, the first a control. | |||
# assmebly | |||
#* assemble a nanostructure with outward- (1) and inward- (2) facing biotinylated oligos | |||
#* incubate assembled nanostructures with fluorescently-labeled streptavidin | |||
#* close the container lids | |||
# baseline protein assay | |||
#* precipitate nanostructures | |||
#* pour off supernatant (containing excess streptavidin) | |||
#* resuspend nanostructures in water | |||
#* measure streptavidin concentrations in each container (pre-digest) | |||
#** ''these are baseline values, and we expect that they should be similar if incubation efficencies for each nanostructure are similar'' | |||
# protease digest | |||
#* digest each nanostructure with protease | |||
#** ''particular protease and protocol must be finalized'' | |||
# measure streptavidin concentrations in each container (post-digest) | |||
#* ''these values should differ if biotinylated streptavidin can still be digested (container 1) and if a closed nanostructure protects its cargo (container 2) | |||
# confirmation of presence of streptavidin | |||
#* treat both containers with DNAse | |||
#* measure streptavidin concentrations | |||
#** ''streptavidin is expected to be bound to biotin, but biotin not bound to oligos'' | |||
#* treat both containers with protease | |||
#* measure streptavidin concentrations | |||
#** ''values should all be close to zero'' | |||
Revision as of 14:17, 19 July 2006
Protection assay
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 μL 2 μM streptavidin with 6 μL 1 μM 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 μL of different dilutions (as per table below), 6 μL of water, and 2 μL of loading dye
- lanes 7-12 were loaded with the same mixtures as lanes 1-6, respectively
| lane | amt of 1x strep-biotin mix (μL) | dilution | H2O added (μL) | final oligo concentration (nM) | amt of oligo in 2 μL diluted mix (fmols) |
| 3 | 2 | 1x | 0 | 500 | 1000 |
| 4 | 1 | 5x | 4 | 100 | 250 |
| 5 | 1 | 10x | 9 | 50 | 100 |
| 6 | 1 | 40x | 39 | 12.5 | 25 |
| 1 | control: 1 μL 2 μM streptavidin (control) to give 2000 fmols streptavidin | ||||
| 2 | control: 2 μL 1 μM oligo (control) to give 2000 fmols oligo | ||||
- 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) [1]
Imaging biotinylated oligos
- goal: show that we can image biotinylated oligos on a PA gel
| lane | non-biotinylated DNA (Lewis' S5) (2 μM) (μL) |
biotyinylated DNA (3.2.7.2b) (1 μM) |
H2O (μL) | Tris-glycine loading dye (10x) (μL) | total amt of DNA (fmols) |
| 1 | 3 | 0 | 5 | 2 | 6000 |
| 2 | 1 | 0 | 7 | 2 | 2000 |
| 3 | 0 | 5 | 3 | 2 | 5000 |
| 4 | 0 | 3 | 5 | 2 | 3000 |
| 5 | 0 | 1 | 7 | 2 | 1000 |
| 6 | 0 | 0.5 | 7.5 | 2 | 5000 |
- run on a 12% native PA gel at 120V for 15 min.
Brainstorming for future assay
Run two experiments in parallel, the first a control.
- assmebly
- assemble a nanostructure with outward- (1) and inward- (2) facing biotinylated oligos
- incubate assembled nanostructures with fluorescently-labeled streptavidin
- close the container lids
- baseline protein assay
- precipitate nanostructures
- pour off supernatant (containing excess streptavidin)
- resuspend nanostructures in water
- measure streptavidin concentrations in each container (pre-digest)
- these are baseline values, and we expect that they should be similar if incubation efficencies for each nanostructure are similar
- protease digest
- digest each nanostructure with protease
- particular protease and protocol must be finalized
- digest each nanostructure with protease
- measure streptavidin concentrations in each container (post-digest)
- these values should differ if biotinylated streptavidin can still be digested (container 1) and if a closed nanostructure protects its cargo (container 2)
- confirmation of presence of streptavidin
- treat both containers with DNAse
- measure streptavidin concentrations
- streptavidin is expected to be bound to biotin, but biotin not bound to oligos
- treat both containers with protease
- measure streptavidin concentrations
- values should all be close to zero
