IGEM:Harvard/2006/DNA nanostructures/Notebook

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
m
m (Protocols)
Line 40: Line 40:
* 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 13:58, 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+
Personal tools