Difference between revisions of "Team:Paris Bettencourt/Protocols/titration-acid-phytic"
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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). | 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). | ||
<img src="https://static.igem.org/mediawiki/2015/8/8f/ParisBttencourt_Calcul_concentration_phytic_acid.png" width="400px"> | <img src="https://static.igem.org/mediawiki/2015/8/8f/ParisBttencourt_Calcul_concentration_phytic_acid.png" width="400px"> | ||
| − | <br>Where :<br> | + | <br><i>Where :</i><br> |
20 = original sample extract volume<br> | 20 = original sample extract volume<br> | ||
F = dilution factor<br> | F = dilution factor<br> | ||
| Line 52: | Line 52: | ||
<b><i>It follows for phosphorous :</i></b> | <b><i>It follows for phosphorous :</i></b> | ||
| + | <img src="https://static.igem.org/mediawiki/2015/d/d4/ParisBettencourt_Calcul_concentration_phosphore_phytic_acid.png" width="400px"> | ||
| + | <b><i>It follows for phytic acid :</i></b> | ||
| + | <img src="https://static.igem.org/mediawiki/2015/4/4b/ParisBettencourt_Calcul_concentration_acide_hytique_phytic_acid.png" width="400px"> | ||
</ul> | </ul> | ||
Revision as of 08:47, 2 September 2015
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 :
<img src="
" width="400px">
<img src="
" width="360px">
<img src="
" width="360px">
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 :<img src="
" width="400px">
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).
<img src="
" width="400px">
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 volumeIt follows for phosphorous : <img src="
" width="400px">
It follows for phytic acid : <img src="
" width="400px">
