IGEM08 How to measure phosphate

From 2008.igem.org

Contents

Reagent preparation

Reagent I

  • Sulfuric acid (H2SO4)
  • Potassium antimonyl tartrate solution
  • Ammonium molybdate solution

Reagent II

  • Ascorbic acid (0.1M): Dissolve 1.76 g ascorbic acid in 100 mL distilled water

Volume ratio between reagents

  • 10X Sulfuric acid (H2SO4)
  • 1X Potassium antimonyl tartrate solution
  • 3X Ammonium molybdate solution
  • 6X ascorbic acid

Dilution of phosphate

start from 10,000 uM stock

start from 1,000 uM stock

Reaction procedure

  • sample 1mL (1000 uL)
  • reagent I (112 uL)
  • reagent II (48uL)
  • STEP 1: mix sample with reagent I
  • STEP 2: vortex the mixture
  • STEP 3: add reagent II (reaction starts!)

Acid concentration and phosphate measurement

NYMU NYMU iGEM08 pi standard curve linear region comparison under different H2SO4 concentration.png

  • pi measure at linear region under different concentration of H2SO4
  • 0.7N slope: 0.0024/uM
  • 2.0N slope: 0.0028/uM
  • 5.0N slope: 0.0017/uM


0.7 N H2SO4

NYMU iGEM08 pi standard curve 0.7N H2SO4.png

2.0 N H2SO4

NYMU iGEM08 pi standard curve 2N H2SO4.png

NYMU iGEM08 pi standard curve 2N H2SO4 (2).png

5.0 N H2SO4

NYMU iGEM08 pi standard curve 5N H2SO4.png

Measure procedure

  • 10 mins reaction time (each sample MUST have equal reaction time)
  • BECKMAN DU-600 at 880nm (圖資大樓6F共儀)
    • Turn VIS 'ON' for at least 5 mins
    • Set parameter 880nm as 'ON'
    • Read blank
    • Read samples
    • Turn VIS 'OFF'

Other methods

  • [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=49192 A continuous spectrophotometric assay for inorganic phosphate and for measuring phosphate release kinetics in biological systems, Proc Natl Acad Sci U S A. 1992]
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