Team:British Columbia/Notebook/Protocols/BiopanningAssay

From 2014.igem.org

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<h1>Biopanning Assay</h1>
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<li>Elution steps
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<li>Treat with elution buffer 1 (EB1) containing 50mM Tris-HCl pH7.5, 250mM NaCl for 15 minutes. Collect Elution.</li>
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<li>Treat with elution buffer 2 (EB2) containing 50mM Tris-HCl pH7.5, 500mM NaCl for 15 minutes. Collect Elution.</li>
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<h4>Tris-buffered saline (TBS) (1X)</h4>
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50 mM Tris-Cl, pH 7.5
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150 mM NaCl
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To prepare, dissolve 6.05 g Tris and 8.76 g NaCl in 800 mL of H2O. Adjust pH to 7.5 with 1 M HCl and make volume up to 1 L with H2O. TBS is stable at 4°C for 3 mo.
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Then add tween.
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<h4>Tris-buffered saline (TBS) (1X)</h4>
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50 mM Tris-Cl, pH 7.5
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<br>
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250 mM NaCl
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<br>
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To prepare, dissolve 6.05 g Tris and 14.61 g NaCl in 800 mL of H2O. Adjust pH to 7.5 with 1 M HCl and make volume up to 1 L with H2O. TBS is stable at 4°C for 3 mo.
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<h4>Tris-buffered saline (TBS) (1X)</h4>
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50 mM Tris-Cl, pH 7.5
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<br>
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500 mM NaCl
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<br>
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To prepare, dissolve 29.22 g Tris and 14.61 g NaCl in 800 mL of H2O. Adjust pH to 7.5 with 1 M HCl and make volume up to 1 L with H2O. TBS is stable at 4°C for 3 mo.
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Latest revision as of 03:50, 18 October 2014

2014 UBC iGEM

Biopanning Assay


Unfixed chalcopyrite:

  1. Pre-sieve chalcopyrite with 40um size cell strainer. Then autoclave by Dry-5 times settings
  2. Incubate chalcopyrite (40um in size, 30mg) in culture of Caulobacter crescentus for 2hrs, shaking at low rpm.
  3. Filter chalcopyrite + Caulobacter crescentus using a 40um Falcon™ Cell Strainers
  4. Washing steps:
    • 1st round of panning: Wash strained chalcopyrite with 5ml 1x Tris-buffered saline (TBS) containing 0.1% (v/v) Tween-20, pH 7.2. Plate cells.
    • 2nd round of panning: Wash strained chalcopyrite with 5ml 1x Tris-buffered saline (TBS) containing 0.2% (v/v) Tween-20, pH 7.2.
    • 3rd round of panning: Wash strained chalcopyrite with 5ml 1x Tris-buffered saline (TBS) containing 0.3% (v/v) Tween-20, pH7.2.
    • 4th round panning: Wash strained chalcopyrite with 5ml 1x Tris-buffered saline (TBS) containing 0.4% (v/v) Tween-20, pH7.2.
    • 5th round panning: Wash strained chalcopyrite with 5ml 1x Tris-buffered saline (TBS) containing 0.5% (v/v) Tween-20, pH7.2
  5. Elution steps
    • Treat with elution buffer 1 (EB1) containing 50mM Tris-HCl pH7.5, 250mM NaCl for 15 minutes. Collect Elution.
    • Treat with elution buffer 2 (EB2) containing 50mM Tris-HCl pH7.5, 500mM NaCl for 15 minutes. Collect Elution.

Tris-buffered saline (TBS) (1X)


50 mM Tris-Cl, pH 7.5
150 mM NaCl
To prepare, dissolve 6.05 g Tris and 8.76 g NaCl in 800 mL of H2O. Adjust pH to 7.5 with 1 M HCl and make volume up to 1 L with H2O. TBS is stable at 4°C for 3 mo.
Then add tween.

Tris-buffered saline (TBS) (1X)


50 mM Tris-Cl, pH 7.5
250 mM NaCl
To prepare, dissolve 6.05 g Tris and 14.61 g NaCl in 800 mL of H2O. Adjust pH to 7.5 with 1 M HCl and make volume up to 1 L with H2O. TBS is stable at 4°C for 3 mo.

Tris-buffered saline (TBS) (1X)


50 mM Tris-Cl, pH 7.5
500 mM NaCl
To prepare, dissolve 29.22 g Tris and 14.61 g NaCl in 800 mL of H2O. Adjust pH to 7.5 with 1 M HCl and make volume up to 1 L with H2O. TBS is stable at 4°C for 3 mo.


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