IGEM:Harvard/2006/DNA nanostructures/Notebook: Difference between revisions

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* 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>
====Protocols====
Potential protocol for a 2 {{ul}} incubation reaction (revised with Dr. Shih's suggestions)
* Reconsitute lyophilized [http://www.sigmaaldrich.com/catalog/search/ProductDetail/SIAL/T6634 bovine thrombin] — ''[[User:Matthewmeisel/Notebook/2006-7-11|done]]''
* In a 0.2 mL PCR tube, mix:
** 0.5 {{ul}} of 4x (not 5x) [[IGEM:Harvard/2006/Stock_solutions#Bock.27s_selection_buffer|Bock's selection buffer]]
** 1.0 {{ul}} of 2 {{um}} aptamers (final concentration: 1.0 {{um}} = 2 pmol)
** 0.5 {{ul}} of 2 {{um}} thrombin (final concentration: 0.5 {{um}} = 1 pmol)
* OR in a 0.2 mL PCR tube, mix:
** 0.5 {{ul}} of 4x (not 5x) [[IGEM:Harvard/2006/Stock_solutions#Bock.27s_selection_buffer|Bock's selection buffer]]
** 0.5 {{ul}} of 2 {{um}} aptamers (final concentration: 0.5 {{um}} = 1 pmol)
** 1.0 {{ul}} of 2 {{um}} thrombin (final concentration: 1.0 {{um}} = 2 pmol)
* Alternative mix: Liu uses 10 pmol of DNA (1 {{ul}} of 10 {{um}}) and varies thrombin amount from 2 pmol (1 {{ul}} of 0.2x thrombin working stock) to 100 pmol (1 {{ul}} of 10x thrombin working stock)
* Incubate at room temperature for 30 min.
* Load onto a non-denaturing polyacrylamide gel (10% to 20% gradient) at 4{{c}}
** Liu runs at 25 mA for 48 h.
[[User:Matthewmeisel|Matthewmeisel]] 11:11, 11 July 2006 (EDT)

Revision as of 11:58, 11 July 2006


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 bands no 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 bands fast 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+
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