Difference between revisions of "Team:Paris Bettencourt/Protocols/titration-acid-phytic"

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<img src="https://static.igem.org/mediawiki/2015/8/83/ParisBettencourt_Tableau_1_dosage_acide_phytic.png" width="400px">
 
<img src="https://static.igem.org/mediawiki/2015/8/83/ParisBettencourt_Tableau_1_dosage_acide_phytic.png" width="400px">
 
       <li><b>Colourimetric determination of phosphorous :</b></li>
 
       <li><b>Colourimetric determination of phosphorous :</b></li>
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<img src="https://static.igem.org/mediawiki/2015/b/ba/ParisBettencourtTab_PHYTIC_ACID_Colourimetric_determination.png" width="400px">
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<img src="https://static.igem.org/mediawiki/2015/b/ba/ParisBettencourtTab_PHYTIC_ACID_Colourimetric_determination.png" width="360px">
 
       <li><b>Preparation of phosphorous calibration curve :</b></li>
 
       <li><b>Preparation of phosphorous calibration curve :</b></li>
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<img src="https://static.igem.org/mediawiki/2015/5/59/ParisBettencourtTab_PHYTIC_ACID_Prep_calibration_curve.png" width="400px">
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<img src="https://static.igem.org/mediawiki/2015/5/59/ParisBettencourtTab_PHYTIC_ACID_Prep_calibration_curve.png" width="360px">
 
       <li><b>Calculation :</b></li>
 
       <li><b>Calculation :</b></li>
 
</ul>
 
</ul>

Revision as of 10:24, 30 August 2015

Titration of phytic acid using Megazyme© Kit
  • Preparation of reagent solutions (not supllied) :
    • Trichloroacetic acid (50% w/v) : 100mL
      • Add 50g of thrichloroacetic to 60mL of distilled water and dissolve. Make to volume (100mL) with distilled water. (Stable for > 6 moths at 4°C)
    • Hydrochloric acid (0.66M) : 1L
      • Add 54.5mL of hydrochloric acid to 945.5mL of distilled wtaer an mix. (Store at room temperature)
    • Sodium hydroxide (0.75M) : 200mL
      • Add 6g of sodium hydroxide pellets to 180mL of distilled water and dissolve. Make to volume (200mL) with distilled water. (Store at room temperature)
    • Phytic acid :
      • Pure phytic acid control sample may be required.
  • Sample extraction :
    • Weigh 1g of sample material.
    • Add 20mL of hydrochloric acid (0.66M).
    • Cover with foil and incubate for a minimum of 3 hours at room temperature.
    • Transfer 1mL of extract to a 1.5mL microcentrifuge tube.
    • Centrifuge 10 minutes at 13,000rpm.
    • Transfer 0.5mL of the resulting extract supernatant to a fresh 1.5mL microfuge tube.
    • Add 0.5mL of sodium hydroxide solution (0.75M) to neutralise.
  • Enzymatic dephosphorylation reaction :

    • <img src="ParisBettencourt_Tableau_1_dosage_acide_phytic.png" width="400px">

  • Colourimetric determination of phosphorous :

    • <img src="ParisBettencourtTab_PHYTIC_ACID_Colourimetric_determination.png" width="360px">

  • Preparation of phosphorous calibration curve :

    • <img src="ParisBettencourtTab_PHYTIC_ACID_Prep_calibration_curve.png" width="360px">

  • Calculation :