IGEM:Harvard/2006/DNA nanostructures/Notebook

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
m
m
Line 1: Line 1:
-
<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}}
-
</div>
+
* Coomassie stain
-
<br style="clear:both">
+
 
-
<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 14:58, 11 July 2006


Contents

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

  • Reconsitute lyophilized bovine thrombindone
  • 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)

Personal tools