Team:Paris Bettencourt/Protocols/titration-acid-phytic
Ferment It Yourself
iGEM Paris-Bettencourt 2O15
- Background
- Design
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Vitamin A Vitamin B2 Vitamin B12 Phytase Riboswitch Differentiation on E. coli Differentiation on S. cerevisiae Manufacturing Idli and Micro-organisms 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).
\[
\begin{align}
C^{\circle} = \frac{mean(M)\times 20 \times F}{10000 \times 1.0 \times \nu} \times \Delta A(phosphorous)
\end{align}
\]
Where :
20 = original sample extract volume
F = dilution factor
10,000 = conversion from µg/g to g/100g
1.0 = wigh of original sample material
v = sample volume It follows for phosphorous :
It follows for phytic acid :
