Team:Calgary/Sandbox/Notebook/Protocols/GelShiftAssay

From 2013.igem.org

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<h2>Reagents and Materials</h2>
<h2>Reagents and Materials</h2>
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<ul>
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<li>Purified protein</li>
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<li>Purified plasmid</li>
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<li>1.3% <a href="https://2013.igem.org/Team:Calgary/Sandbox/Notebook/Protocols/AgaroseGelElectrophoresis" target="_blank">agarose gel</a></li>
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</ul>
<p class="noIndent"><b>10x Buffer</b></p>
<p class="noIndent"><b>10x Buffer</b></p>
<ul>
<ul>

Revision as of 20:32, 22 September 2013

Gel Shift Assay

Reagents and Materials

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
  • 1.3% agarose gel
  • DNA of interest
  • Purified protein sample

Protocol

  1. Perform a Bradford Assay to the protein concentration
  2. Add 2µL of 10X Buffer, 55pM of DNA, 0.01 to 2500nM of Protein, and fill up to 20µL with ddH2O. Mix thoroughly
  3. Incubate for 1 hour at room temperature in the dark
  4. Place at 4°C for 30 minutes
  5. Run on a 1.3% agarose gel at 50V

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