Team:Calgary/Notebook/Protocols/FunctionalityAssayOnNitrocellulose

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<h1>Functionality Assay On Nitrocellulose</h1>
<h1>Functionality Assay On Nitrocellulose</h1>
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<h2> Reagents and Materials</h2>
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<ul>
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<li>Nitrocellulose paper</li>
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<li>Purified protein</li>
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<li>FAM-labelled double stranded DNA</li>
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<li><a href="https://2013.igem.org/Team:Calgary/Notebook/Protocols/GelShiftAssay
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" ><span class="blue"><b>1x TALE binding buffer
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</b></span></a></a></li>
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<li>TBST buffer</li>
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<li>5% skimmed milk in TBST buffer</li>
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</ul>
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<h2>Protocol<//h2>
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<ol>
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<li>Cut thin strips of nitrocellulose paper; thin enough to fit into a 2mL tube</li>
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<li>Blot enough protein on the strip</li>
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<li>put the strips in a container and block them with 5% skimmed milk in TBST buffer for at least 30 minutes</li>
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<li>Discard the milk and wash the strips 3 times with TBST buffer</li>
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<li>make a 1.6 mM solution with the FAM-lablled DNA in 2mL tubes. The FAM-lablled DNA must be protected from light. </li>
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<li>Insert the strips into the DNA tubes and let them soak for appropriate length of time</li>
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<li>Wash the strips twice with 1x TALE-binding buffer.</li>
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<li>Image the strips.</li>
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</ol>
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</ul>
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Latest revision as of 01:43, 29 October 2013

Functionality Assay On Nitrocellulose

Reagents and Materials

  • Nitrocellulose paper
  • Purified protein
  • FAM-labelled double stranded DNA
  • 1x TALE binding buffer
  • TBST buffer
  • 5% skimmed milk in TBST buffer

Protocol
  1. Cut thin strips of nitrocellulose paper; thin enough to fit into a 2mL tube
  2. Blot enough protein on the strip
  3. put the strips in a container and block them with 5% skimmed milk in TBST buffer for at least 30 minutes
  4. Discard the milk and wash the strips 3 times with TBST buffer
  5. make a 1.6 mM solution with the FAM-lablled DNA in 2mL tubes. The FAM-lablled DNA must be protected from light.
  6. Insert the strips into the DNA tubes and let them soak for appropriate length of time
  7. Wash the strips twice with 1x TALE-binding buffer.
  8. Image the strips.

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