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

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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
12% native PAGE. Lower detection threshold of streptavidin is 500 fmols (lane 3).
  • 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
12% native PAGE. Lower detection limit for biotinylated oligos is 3000 fmols (lane 4).
  • 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
    • assemble a nanostructure with outward- (1) and inward- (2) facing biotinylated oligos
    • incubate assembled nanostructures with fluorescently-labeled streptavidin
    • close the container lids
  2. 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
  3. protease digest
    • digest each nanostructure with protease
      • particular protease and protocol must be finalized
  4. 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)
  5. 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

Container 4.0

Reagents (each expt in respective 0.2 mL PCR tube)

Experiment Scaffold Oligos Folding buffer dH2O total volume
-latches
-aptamers
9 μL p7308 16 μL xxx (250 nm) 4 μL 10x 11 μL 40 μL
+latch1
-aptamers
9 μL p7308 16 μL xxx (250 nm) 4 μL 10x 11 μL 40 μL
+latch2
-aptamers
9 μL p7308 16 μL xxx (250 nm) 4 μL 10x 11 μL 40 μL
6 hb 9 μL p7308 4 μL 6hb.v5 (0.99 μM) 4 μL 10x 23 μL 40 μL
-oligos 9 μL p7308 - 4 μL 10x 27 μL 40 μL
-scaffold - 16 μL xxx (250 nm) 4 μL 10x 20 μL 40 μL

Annealing protocol

  • start at 80[[:Category:{{{1}}}|{{{1}}}]]
  • 60 cycles: wait 2 minutes, decrease 1[[:Category:{{{1}}}|{{{1}}}]]
  • hold at 4[[:Category:{{{1}}}|{{{1}}}]]

Gel analysis

  • 2% agarose gel supplemented to 10 mM MgCl2 and with 3 μL 10 mg/mL EtBr (100 mL gel)
  • run in 1x TBE supplemented to 10 mM MgCl2
  • 45 min at 130 V
Lane Contents Loading Buffer (10x TBE/glycerol)
1 1kb DNA ladder (10 μL) 1.1 μL
2 (-) 1.1 μL
3 Ib (10 μL) 1.1 μL
4 4.0 -latches -aptamers (10 μL) 1.1 μL
5 4.0 +latch1 -aptamers (10 μL) 1.1 μL
6 4.0 +latch2 -aptamers (10 μL) 1.1 μL
7 6hb (10 μL) 1.1 μL
8 4.0 -oligos (10 μL) 1.1 μL
9 4.0 -scaffold 1.1 μL
  • results
2% agarose gel electrophoresis. All folded structures run slower than scaffold alone.