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

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Contents

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)
321x05001000
415x4100250
5110x950100
6140x3912.525
1control: 1 μL 2 μM streptavidin (control) to give 2000 fmols streptavidin
2control: 2 μL 1 μM oligo (control) to give 2000 fmols oligo
12% native PAGE. Lower detection threshold of streptavidin is 500 fmols (lane 3).
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)
130526000
210722000
305325000
403523000
501721000
600.57.525000
12% native PAGE. Lower detection limit for biotinylated oligos is 3000 fmols (lane 4).
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 p730816 μL xxx (250 nm)4 μL 10x11 μL40 μL
+latch1
-aptamers
9 μL p730816 μL xxx (250 nm)4 μL 10x11 μL40 μL
+latch2
-aptamers
9 μL p730816 μL xxx (250 nm)4 μL 10x11 μL40 μL
6 hb9 μL p73084 μL 6hb.v5 (0.99 μM)4 μL 10x23 μL40 μL
-oligos9 μL p7308-4 μL 10x27 μL40 μL
-scaffold-16 μL xxx (250 nm)4 μL 10x20 μL40 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)
11kb DNA ladder (10 μL)1.1 μL
2(-)1.1 μL
3Ib (10 μL)1.1 μL
44.0 -latches -aptamers (10 μL)1.1 μL
54.0 +latch1 -aptamers (10 μL)1.1 μL
64.0 +latch2 -aptamers (10 μL)1.1 μL
76hb (10 μL)1.1 μL
84.0 -oligos (10 μL)1.1 μL
94.0 -scaffold1.1 μL
  • results
2% agarose gel electrophoresis. All folded structures run slower than scaffold alone.
2% agarose gel electrophoresis. All folded structures run slower than scaffold alone.
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