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

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     <td><b>Titration of phytc acid using Megazyme© Kit</b><br />
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     <td><b>Titration of phytic acid using Megazyme© Kit</b><br />
 
     <ul>
 
     <ul>
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       <li>Preparation of reagent solutions (not supllied) :</li>
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       <li><b>Preparation of reagent solutions (not supllied) :</b></li>
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      <li>Add 200µL Buffer AL. Mix thoroughly by vortexing.</li>
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          <ul>
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      <li>Add 200µL ethanol (96%). Mix thoroughly by vortexing.</li>
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          <li><i><b>Trichloroacetic acid  (50% w/v) : 100mL</b></i></li>
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      <li>Pipet the mixture into a DNeasy Mini spin column placed in a 2mL collection tube. Centrifuge at 6000g (8000rpm) for 1 minute. Discard the flow-through and collection tube.</li>
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          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)</li>
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       <li>Place the spin column in a new 2mL collection tube. Add 500µL Buffer AW1. Centrifuge for 1 minute at 6000g. Discard the flow-through and collection tube.</li>
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          <li><i><b>Hydrochloric acid (0.66M) : 1L</b></i></li>
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      <li>Place the spin column in a new 2mL collection tube. Add 500µL Buffer AW2. Centrifuge for 3 minutes at 20,000g. Discard the flow-through and collection tube.</li>
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          Add 54.5mL of hydrochloric acid to 945.5mL of distilled wtaer an mix. (Store at room temperature)</li>
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      <li>Transfer the spin column to a new 1.5mL or 2mL microcentrifuge tube.</li>
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          <li><i><b>Sodium hydroxide (0.75M) : 200mL</b></i></li>
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       <li>Elute the DNA by adding 200µL Buffer AE or sterile water to the center of the spin column membrane. Incubate for 1 minute at room temperature. Centrifuge for 1 minute at 6000g.</li>
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          Add 6g of sodium hydroxide pellets to 180mL of distilled water and dissolve. Make to volume (200mL) with distilled water. (Store at room temperature)</li>
−
      <li>Repeat this step for increased DNA yield.</li>
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          <li><i><b>Phytic acid :</b></i></li>
−
    </ul>
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          Pure phytic acid control sample may be required.</li>
 +
          </ul>
 +
       <li><b>Sample extraction :</b></li>
 +
          <ul>
 +
          <li>Weigh 1g of sample material.</li>
 +
          <li>Add 20mL of hydrochloric acid (0.66M).</li>
 +
          <li>Cover with foil and incubate for a minimum of 3 hours at room temperature.</li>
 +
          <li>Transfer 1mL of extract to a 1.5mL microcentrifuge tube.</li>
 +
          <li>Centrifuge 10 minutes at 13,000rpm.</li>
 +
          <li>Transfer 0.5mL of the resulting extract supernatant to a fresh 1.5mL microfuge tube.</li>
 +
          <li>Add 0.5mL of sodium hydroxide solution (0.75M) to neutralise.</li>
 +
          </ul>
 +
       <li><b>Enzymatic dephosphorylation reaction :</b></li>
 +
<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>
 +
<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>
 +
<img src="https://static.igem.org/mediawiki/2015/5/59/ParisBettencourtTab_PHYTIC_ACID_Prep_calibration_curve.png" width="360px">
 +
      <li><b>Calculation :</b></li>
 +
<ul>
 +
      <li><b>Phosphorous calibration curve :</b></li>
 +
      Determine the absorbance of each phosphorous standard. Substract the absorbance of STD0 from the absorbance of the others STD, therby obtaining ΔA(phosphorous).
 +
      Calculate M as follows, for each standard:
 +
\[
 +
\begin{align}
 +
M = \frac{P(\mu g)}{\Delta A (phosphorous)}
 +
\end{align}
 +
\]
 +
      Use "Mean M" to calculate the phosphorous content of test samples.<br>
 +
      <li><b>Phosphorous / phytic acid content :</b></li>
 +
      Determine the absorbance of the both "Free Phosphorous" and "Total Phosphorous". Substract the absorbance of the "Free Phosphorous" sample from the absorbance of the "Total Phosphorous" sample. Obtaining ΔA(phosphorous).
 +
\[
 +
\begin{align}
 +
C^{o} = \frac{mean(M)\times 20 \times F}{10000 \times 1.0 \times \nu} \times \Delta A(phosphorous)
 +
\end{align}
 +
\]
 +
<br><i>Where :</i><br>
 +
\(20\) = original sample extract volume<br>
 +
\(F\) = dilution factor<br>
 +
\(10000\) = conversion from µg/g to g/100g<br>
 +
\(1.0\) = wigh of original sample material<br>
 +
\(\nu\) = sample volume
 +
 
 +
<br><br>
 +
<b><i>It follows for phosphorous :</i></b><br>
 +
\[
 +
\begin{align}
 +
C^{o} ~= \frac{mean(M)\times 20 \times 55.6}{10000 \times 1.0 \times \nu} \times \Delta A(phosphorous)\\
 +
~= mean(M) \times 0.1112 \times \Delta A(phosphorous)
 +
\end{align}
 +
\]
 +
 
 +
<b><i>It follows for phytic acid :</i></b><br>
 +
\[
 +
\begin{align}
 +
C^{o} = \frac{phosphorus}{0.282}
 +
\end{align}
 +
\]
 +
 
 +
</ul>
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</ul>
 
     </td>
 
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Latest revision as of 16:40, 2 September 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 :
  • Colourimetric determination of phosphorous :
  • Preparation of phosphorous calibration curve :
  • Calculation :
    • Phosphorous calibration curve :
      • Determine the absorbance of each phosphorous standard. Substract the absorbance of STD0 from the absorbance of the others STD, therby obtaining ΔA(phosphorous). Calculate M as follows, for each standard: \[ \begin{align} M = \frac{P(\mu g)}{\Delta A (phosphorous)} \end{align} \] Use "Mean M" to calculate the phosphorous content of test samples.
    • Phosphorous / phytic acid content :
      • Determine the absorbance of the both "Free Phosphorous" and "Total Phosphorous". Substract the absorbance of the "Free Phosphorous" sample from the absorbance of the "Total Phosphorous" sample. Obtaining ΔA(phosphorous). \[ \begin{align} C^{o} = \frac{mean(M)\times 20 \times F}{10000 \times 1.0 \times \nu} \times \Delta A(phosphorous) \end{align} \]
      Where :
      \(20\) = original sample extract volume
      \(F\) = dilution factor
      \(10000\) = conversion from µg/g to g/100g
      \(1.0\) = wigh of original sample material
      \(\nu\) = sample volume

      It follows for phosphorous :
      \[ \begin{align} C^{o} ~= \frac{mean(M)\times 20 \times 55.6}{10000 \times 1.0 \times \nu} \times \Delta A(phosphorous)\\ ~= mean(M) \times 0.1112 \times \Delta A(phosphorous) \end{align} \] It follows for phytic acid :
      \[ \begin{align} C^{o} = \frac{phosphorus}{0.282} \end{align} \]