Team:Calgary/Notebook/Protocols/GelShiftAssay

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<li>50 mM MgCl2</li>
<li>50 mM MgCl2</li>
<li>2mM EDTA</li>
<li>2mM EDTA</li>
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<li>Fill up to desired volume with water</li>
+
<li>ddH<sub>2</sub>O (deionized water)</li>
<li>pH = 7.5</li>
<li>pH = 7.5</li>
</ul>
</ul>

Revision as of 00:32, 28 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
  • ddH2O (deionized water)
  • pH = 7.5

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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