"
Team:Calgary/Sandbox/Notebook/Protocols/GelShiftAssay
From 2013.igem.org
(Difference between revisions)
Azzucoloto (Talk | contribs) |
|||
| (One intermediate revision not shown) | |||
| Line 5: | Line 5: | ||
<h2>Reagents and Materials</h2> | <h2>Reagents and Materials</h2> | ||
| + | <ul> | ||
| + | <li>1.3% <a href="https://2013.igem.org/Team:Calgary/Sandbox/Notebook/Protocols/AgaroseGelElectrophoresis">agarose gel</a></li> | ||
| + | <li>DNA of interest</li> | ||
| + | <li>Purified protein sample</li> | ||
| + | </ul> | ||
<p class="noIndent"><b>10x Buffer</b></p> | <p class="noIndent"><b>10x Buffer</b></p> | ||
<ul> | <ul> | ||
| Line 17: | Line 22: | ||
<li>Fill up to desired volume with water</li> | <li>Fill up to desired volume with water</li> | ||
<li>pH = 7.5</li> | <li>pH = 7.5</li> | ||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
</ul> | </ul> | ||
<h2>Protocol</h2> | <h2>Protocol</h2> | ||
Latest revision as of 20:32, 22 September 2013
Gel Shift Assay
Reagents and Materials
- 1.3% agarose gel
- DNA of interest
- Purified protein sample
10x Buffer
- 120mM Tris Cl
- 600mM KCl
- 20mM DTT
- 0.5% NP-40
- 1mg/mL BSA
- 50% Glycerol
- 50 mM MgCl2
- 2mM EDTA
- Fill up to desired volume with water
- pH = 7.5
Protocol
- Perform a Bradford Assay to the protein concentration
- Add 2µL of 10X Buffer, 55pM of DNA, 0.01 to 2500nM of Protein, and fill up to 20µL with ddH2O. Mix thoroughly
- Incubate for 1 hour at room temperature in the dark
- Place at 4°C for 30 minutes
- Run on a 1.3% agarose gel at 50V
