Team:Paris Bettencourt/Protocols/titration-acid-phytic
Ferment It Yourself
iGEM Paris-Bettencourt 2O15
- Background
- Design
-
-
-
-
-
-
-
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 :
<img src="
" width="400px">
<img src="
" width="400px">
<img src="
" width="400px">
