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

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<div class="tabs-blue">
 
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<ul>
 
<ul>
<li>[[IGEM:Harvard/2006/DNA_nanostructures|1. Project Overview]]</li>
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<li>[[IGEM:Harvard/2006/DNA nanostructures|Project Overview]]</li>
<li>[[IGEM:Harvard/2006/DNA_Nanostructures/Designs|2. Designs]]</li>
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<li>[[IGEM:Harvard/2006/DNA_nanostructures/Designs|Designs]]</li>
<li id="current">[[IGEM:Harvard/2006/DNA_nanostructures/Notebook|3. Notebook]]</li>
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<li id="current">[[IGEM:Harvard/2006/DNA_nanostructures/Notebook|Notebook]]</li>
<li>[[IGEM:Harvard/2006/DNA_nanostructures/Protocols|4. Protocols]]</li>
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<li>[[IGEM:Harvard/2006/DNA_nanostructures/Protocols|Protocols]]</li>
<li>[[IGEM:Harvard/2006/DNA_nanostructures/Literature|4. Literature]]</li>
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<li>[[IGEM:Harvard/2006/DNA_nanostructures/Presentations|Presentations]]</li>
 +
<li>[[IGEM:Harvard/2006/DNA_nanostructures/Literature|Literature]]</li>
 
</ul>
 
</ul>
 
</div>
 
</div>
 
<br style="clear:both">
 
<br style="clear:both">
<div class="tabcontent">
+
 
 +
==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 {{um}}, DNA at 2 {{um}}
 +
* Coomassie stain
 +
 
 +
====Experiments====
 +
{| {{table}}
 +
| align="center" style="background:#f0f0f0;"|number
 +
| align="center" style="background:#f0f0f0;"|thrombin
 +
| align="center" style="background:#f0f0f0;"|aptamer
 +
| align="center" style="background:#f0f0f0;"|nanotube
 +
| align="center" style="background:#f0f0f0;"|DNA-stained prediction
 +
| align="center" style="background:#f0f0f0;"|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 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 PAGE buffer: 1x TAE/Mg<sup>2+</sup>

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+