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

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<div class="tabs-blue">
{{IGEM H06 DNA nano navbar}}
<ul>
 
<li>[[IGEM:Harvard/2006/DNA_nanostructures|1. Project Overview]]</li>
==Thrombin-aptamer experiments==
<li>[[IGEM:Harvard/2006/DNA_Nanostructures/Designs|2. Designs]]</li>
 
<li id="current">[[IGEM:Harvard/2006/DNA_nanostructures/Notebook|3. Notebook]]</li>
====Questions / procedures====
<li>[[IGEM:Harvard/2006/DNA_nanostructures/Protocols|4. Protocols]]</li>
* what percent gel? 10% to 20% polyacrylamide gels, no SDS (but would make for a good control)
<li>[[IGEM:Harvard/2006/DNA_nanostructures/Literature|5. Literature]]</li>
* what incubation conditions?
</ul>
* how much protein and DNA? protein at 1 {{um}}, DNA at 2 {{um}}
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* Coomassie stain
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<div class="tabcontent">
====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>
 
====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+

Protocols

Potential protocol for a 2 μL incubation reaction (revised with Dr. Shih's suggestions)

  • Reconsitute lyophilized bovine thrombin — done
  • In a 0.2 mL PCR tube, mix:
    • 0.5 μL of 4x (not 5x) Bock's selection buffer
    • 1.0 μL of 2 μM aptamers (final concentration: 1.0 μM = 2 pmol)
    • 0.5 μL of 2 μM thrombin (final concentration: 0.5 μM = 1 pmol)
  • OR in a 0.2 mL PCR tube, mix:
    • 0.5 μL of 4x (not 5x) Bock's selection buffer
    • 0.5 μL of 2 μM aptamers (final concentration: 0.5 μM = 1 pmol)
    • 1.0 μL of 2 μM thrombin (final concentration: 1.0 μM = 2 pmol)
  • Alternative mix: Liu uses 10 pmol of DNA (1 μL of 10 μM) and varies thrombin amount from 2 pmol (1 μL of 0.2x thrombin working stock) to 100 pmol (1 μL 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[[:Category:{{{1}}}|{{{1}}}]]
    • Liu runs at 25 mA for 48 h.

Matthewmeisel 11:11, 11 July 2006 (EDT)

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