IGEM:Harvard/2006/DNA nanostructures

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==Thrombin-aptamer experiments==
==Thrombin-aptamer experiments==
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====Notes====
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====Questions / procedures====
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Questions / procedures
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* what percent gel? 10% to 20% polyacrylamide gels, no SDS (but would make for a good control)
* what percent gel? 10% to 20% polyacrylamide gels, no SDS (but would make for a good control)
* what incubation conditions?
* what incubation conditions?
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* Coomassie stain
* Coomassie stain
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Experiments
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====Experiments====
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Buffers
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====Buffers====
* Macaya's and Bock's selection buffer: 20 mM Tris-acetate, pH 7.4, 140 mM NaCl, 5 mM KCl, 1 mM CaCl<sub>2</sub>, 1 mM MgCl<sub>2</sub>
* Macaya's and Bock's selection buffer: 20 mM Tris-acetate, pH 7.4, 140 mM NaCl, 5 mM KCl, 1 mM CaCl<sub>2</sub>, 1 mM MgCl<sub>2</sub>
* Liu's incubation buffer: 40 mM Tris, 20 mM CH<sub>3</sub>COOH, 2mM EDTA, 12.5 mM (CH<sub>3</sub>COO)<sub>2</sub>Mg, pH 8.0
* Liu's incubation buffer: 40 mM Tris, 20 mM CH<sub>3</sub>COOH, 2mM EDTA, 12.5 mM (CH<sub>3</sub>COO)<sub>2</sub>Mg, pH 8.0
* Liu's PAGE buffer: 1x TAE/Mg<sup>2+</sup>
* Liu's PAGE buffer: 1x TAE/Mg<sup>2+</sup>
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Potential procedure for 10 {{ul}} incubation reaction
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====Protocols====
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* In 1 0.2 mL PCR tube, mix:
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Potential protocol for 10 {{ul}} incubation reaction
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* In a 0.2 mL PCR tube, mix:
** 2 {{ul}} of 5x Bock's selection buffer
** 2 {{ul}} of 5x Bock's selection buffer
** 2 {{ul}} of 10 {{um}} scaffold DNA (final concentration: 2 {{um}} = 20 pmol)
** 2 {{ul}} of 10 {{um}} scaffold DNA (final concentration: 2 {{um}} = 20 pmol)
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* Load onto a non-denaturing polyacrylamide gel (10% to 20% gradient)
* Load onto a non-denaturing polyacrylamide gel (10% to 20% gradient)
** Liu runs at 25 mA for 48 h.
** Liu runs at 25 mA for 48 h.
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[[User:Matthewmeisel|Matthewmeisel]] 17:13, 10 July 2006 (EDT)
====Bibliography====
====Bibliography====

Revision as of 17:13, 10 July 2006

Contents

Project Overview

  • Our goal is to to design and implement molecular containers, which can be dynamically opened and closed by an external stimulus.
  • The containers will be implemented as DNA nanostructures, which afford a significant degree of positional control and chemical versatility.
  • As an initial proof-of-concept, we plan to use our DNA containers to demonstrate controllable activation ("delivery") of anti-thrombin aptamers.
  • We expect that molecular containers could have several interesting scientific and clinical applications, such as
    • Drug and gene delivery
    • Bio-marker scavenging (early detection of biomarkers)
    • Directed evolution (compartmentalized selections)
    • Using multiplexing for combinatorial chemical synthesis
    • Capture and stabilization of multiprotein complexes
    • Protein folding (chaperones)
    • Cell sorting

Container Specs

Image:iGEM_harv06_mattspecs.gif

Container Designs

Latch Designs

Coding

Existing code

Thrombin-aptamer experiments

Questions / procedures

  • what percent gel? 10% to 20% polyacrylamide gels, no SDS (but would make for a good control)
  • what incubation conditions?
  • how much protein and DNA? protein at 1 μM, DNA at 2 μM
  • Coomassie stain

Experiments

number thrombin aptamer nanotube DNA-stained prediction protein-stained prediction
0---no bandsno bands
1--+slow band (nanotube)no bands
2-+-fast band (aptamer)no bands
3-++slow band (aptamer-nanotube), traces of fast band (aptamer)no bands
4+--no bandsfast band (thrombin)
5+-+slow band (nanotube)fast band (thrombin)
6++-medium band (aptamer-thrombin), fast band (aptamer)medium band (aptamer-thrombin), traces of fast band (thrombin)
7+++very slow band (thrombin-aptamer-nanotube), slow band (aptamer-nantotube), traces of fast band (aptamer)very slow band (thrombin-aptamer-nanotube), medium band (aptamer-thrombin), traces of fast band (thrombin)

Buffers

  • Macaya's and Bock's selection buffer: 20 mM Tris-acetate, pH 7.4, 140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2
  • Liu's incubation buffer: 40 mM Tris, 20 mM CH3COOH, 2mM EDTA, 12.5 mM (CH3COO)2Mg, pH 8.0
  • Liu's PAGE buffer: 1x TAE/Mg2+

Protocols

Potential protocol for 10 μL incubation reaction

  • In a 0.2 mL PCR tube, mix:
    • 2 μL of 5x Bock's selection buffer
    • 2 μL of 10 μM scaffold DNA (final concentration: 2 μM = 20 pmol)
    • 2 μL of 10 μM oligos (final concentration: 2 μM = 20 pmol)
    • 2 μL of 10 μM aptamers (final concentration: 2 μM = 20 pmol)
    • 2 μL of 5 μM thrombin (final concentration: 1 μM = 10 pmol)
  • Alternative mix: Liu uses 10 pmol (1 μL of 10 μM) and varies thrombin from 2 pmol (1 μL of
  • Incubate at room temperature for 30 min.
  • Load onto a non-denaturing polyacrylamide gel (10% to 20% gradient)
    • Liu runs at 25 mA for 48 h.

Matthewmeisel 17:13, 10 July 2006 (EDT)

Bibliography

  1. Schultze P, Macaya RF, and Feigon J. . pmid:8107090. PubMed HubMed [tha1]
  2. Liu Y, Lin C, Li H, and Yan H. . pmid:15945116. PubMed HubMed [tha2]
  3. Li WX, Kaplan AV, Grant GW, Toole JJ, and Leung LL. . pmid:8298130. PubMed HubMed [tha3]
  4. Bock LC, Griffin LC, Latham JA, Vermaas EH, and Toole JJ. . pmid:1741036. PubMed HubMed [tha4]
  5. Macaya RF, Schultze P, Smith FW, Roe JA, and Feigon J. . pmid:8475124. PubMed HubMed [tha5]
All Medline abstracts: PubMed HubMed

Presentations

Most recent (Week 3)

Week 2: Original proposal

Working Team Members

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