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

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{{IGEM H06 DNA nano navbar}}
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<div class="tabs-blue">
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<ul>
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<li>[[IGEM:Harvard/2006/DNA nanostructures|Project Overview]]</li>
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<li>[[IGEM:Harvard/2006/DNA_nanostructures/Designs|Designs]]</li>
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<li id="current">[[IGEM:Harvard/2006/DNA_nanostructures/Notebook|Notebook]]</li>
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<li>[[IGEM:Harvard/2006/DNA_nanostructures/Protocols|Protocols]]</li>
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<li>[[IGEM:Harvard/2006/DNA_nanostructures/Presentations|Presentations]]</li>
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<li>[[IGEM:Harvard/2006/DNA_nanostructures/Literature|Literature]]</li>
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</ul>
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</div>
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<br style="clear:both">
  
==Thrombin-aptamer experiments==
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==Daily notebook==
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<calendar>
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name=IGEM:Harvard/2006/DNA_nanostructures/Notebook
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date=2006/07/01
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view=threemonths
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format=%name/%year-%month-%day
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weekstart=7
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</calendar>
 +
 
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<calendar>
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name=IGEM:Harvard/2006/DNA_nanostructures/Notebook
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date=2006/10/01
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view=threemonths
 +
format=%name/%year-%month-%day
 +
weekstart=7
 +
</calendar>
 +
 
 +
==General Notes==
 +
*'''Stock Oligos''': reconstituted (50uM for non-biotinylated oligos, 200uM for biotinylated) oligos in wells or tubes
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*'''Pre-working Stocks''': mixtures of 10uM of each oligo needed for a "class" of oligos (ex. core, barrel, lid1, lid2); for non-biot oligos, this requires 10uL of each of the necessary stock oligos, for biot-oligos, this requires 2.5uL of stock oligos + 7.5uL dH2O
 +
*'''Working Stocks''': mixtures of however-many-oligos-are-in-each-class uL (ex. if there are 94 oligos in the core class of 3.2's design, use 94uL of the core pre-working stock), diluted to a total of 200uL of solution (each individual oligo will be at 250nM)
 +
 
 +
==Brainstorming and future projects==
 +
 
 +
===Thrombin-aptamer experiments===
  
 
====Questions / procedures====
 
====Questions / procedures====
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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)
 

Latest revision as of 17:10, 5 September 2006


Daily notebook

<calendar> name=IGEM:Harvard/2006/DNA_nanostructures/Notebook date=2006/07/01 view=threemonths format=%name/%year-%month-%day weekstart=7 </calendar>

<calendar> name=IGEM:Harvard/2006/DNA_nanostructures/Notebook date=2006/10/01 view=threemonths format=%name/%year-%month-%day weekstart=7 </calendar>

General Notes

  • Stock Oligos: reconstituted (50uM for non-biotinylated oligos, 200uM for biotinylated) oligos in wells or tubes
  • Pre-working Stocks: mixtures of 10uM of each oligo needed for a "class" of oligos (ex. core, barrel, lid1, lid2); for non-biot oligos, this requires 10uL of each of the necessary stock oligos, for biot-oligos, this requires 2.5uL of stock oligos + 7.5uL dH2O
  • Working Stocks: mixtures of however-many-oligos-are-in-each-class uL (ex. if there are 94 oligos in the core class of 3.2's design, use 94uL of the core pre-working stock), diluted to a total of 200uL of solution (each individual oligo will be at 250nM)

Brainstorming and future projects

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+