Team:NYMU-Taipei/Project/Phosphate/Experiments/Pi measure

From 2008.igem.org

Contents

Standard Curve in ddH2O

7/23, 2008 PM 4:04

  • 100, 200, 300, 400, 500 uM pi
  • reaction time: 10 mins
  • 5N H2SO4

Pi standard curve 20080723.png

  • 0.8075 (100 uM)
  • 0.6211 (200 uM)
  • 0.8208 (300 uM)
  • 1.0787 (400 uM)
  • 1.3167 (500 uM)
  • overall (point 1-5) R2 is 0.8608
  • points 2-5 R2 is 0.9973
  • something wrong with the first point (pi = 100uM) measure


7/30, 2008 PM 8:00

  • 500, 1000, 1500, 2000, 2500, 3000 uM pi
  • reaction time: 11 mins (the timing of measure is also important)
  • 5N H2SO4
  • Because the high [pi] is out of the linear range, we have to dilute the samples into linear region
  • standard samples are diluted from 10,000 uM stock
  • The dilution procedure are:
    • extract 30 uL from 10,000 uM stock + 70 uL ddH2O, and add 900 uL ddH2O to 1 C.C. (3000uM, 10X dilution)
    • extract 25 uL from 10,000 uM stock + 75 uL ddH2O, and add 900 uL ddH2O to 1 C.C. (2500uM, 10X dilution)
    • extract 20 uL from 10,000 uM stock + 80 uL ddH2O, and add 900 uL ddH2O to 1 C.C. (2000uM, 10X dilution)
    • extract 15 uL from 10,000 uM stock + 85 uL ddH2O, and add 900 uL ddH2O to 1 C.C. (1500uM, 10X dilution)
    • extract 10 uL from 10,000 uM stock + 90 uL ddH2O, and add 900 uL ddH2O to 1 C.C. (1000uM, 10X dilution)
    • extract 05 uL from 10,000 uM stock + 95 uL ddH2O, and add 900 uL ddH2O to 1 C.C. (0500uM, 10X dilution)
    • However, the precision of this dilution procedure is based on the how precise u can extract from 10,000 uM stock
  • Results

NYMU NYMU iGEM08 pi standard curve 20080730 PM0800.png

  • 1.8472 (3000 uM, 10X dilution)
  • 1.5183 (2500 uM, 10X dilution)
  • 1.8385 (2000 uM, 10X dilution)
  • 1.7027 (1500 uM, 10X dilution)
  • 1.8300 (1000 uM, 10X dilution)
  • 1.7248 (0500 uM, 10X dilution)
  • overall R2 is -0.0792


7/30, 2008 PM 9:48

  • 100, 200, 300, 400, 500 uM pi
  • reaction time 10 mins
  • 5N H2SO4
  • Result

NYMU NYMU iGEM08 pi standard curve 20080730 PM0948.png

  • 1.4077 (100 uM)
  • 1.4355 (200 uM)
  • 1.4991 (300 uM)
  • 1.4894 (400 uM)
  • 1.5477 (500 uM)
  • overall R2 is 0.9542


7/30, 2008 PM 10:00

  • 500, 1000, 1500, 2000, 2500 uM pi (10X dilution)
  • reaction time 10 mins
  • 5N H2SO4
  • Result

NYMU NYMU iGEM08 pi standard curve 20080730 PM1000.png

  • 0.9445 (0500 uM)
  • 1.0810 (1000 uM)
  • 1.0153 (1500 uM)
  • 1.1281 (2000 uM)
  • 1.1631 (2500 uM)
  • overall R2 is 0.8721


8/14, 2008 Trial #1

  • 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 uM pi
  • 2 replicates
  • test on Thermo Scientific Varioskan Flash
  • reaction time 10 mins

NYMU IGEM08 pi standard curve 5N H2SO4 20080814 trial 1 10mins.png

  • blank (0.350265, 0.374877)
  • 50 uM (1.15599, 1.16707)
  • 60 uM (1.31682, 1.38306)
  • 70 uM (0.832704, 0.904693)
  • 80 uM (1.60057, 1.51395)
  • 90 uM (1.44425, 1.49588)
  • 100 uM (1.24785, 1.36892)
  • 200 uM (1.07019, 1.26758)
  • 300 uM (1.41406, 1.58698)
  • 400 uM (0.805028, 0.797246)
  • 500 uM (0.606312, 0.630793)
  • R2 (-0.2393, -0.2138)


8/14, 2008 Trial #2

  • 60, 70, 80, 90, 100, 200, 300, 400, 500 uM pi
  • 2 replicates
  • test on Thermo Scientific Varioskan Flash
  • reaction time 10 mins

NYMU IGEM08 pi standard curve 5N H2SO4 20080814 trial 2 10mins.png

  • blank (0.391602, 0.377879)
  • 60 uM (1.18088, 1.24429)
  • 70 uM (1.69246, 1.71659)
  • 80 uM (1.06502, 1.22468)
  • 90 uM (1.26321, 1.28261)
  • 100 uM (1.27818, 1.22613)
  • 200 uM (1.1761, 1.19522)
  • 300 uM (0.43635, 0.463949)
  • 400 uM (0.629852, 0.632093)
  • 500 uM (0.510317, 0.534434)
  • R2 (-0.5205, -0.5239)
  • reaction time 20 mins

NYMU IGEM08 pi standard curve 5N H2SO4 20080814 trial 2 20mins.png

  • blank (0.874747, 0.843729)
  • 60 uM (2.21735, 2.34065)
  • 70 uM (2.80967, 2.84848)
  • 80 uM (2.07216, 2.36317)
  • 90 uM (2.27881, 2.30526)
  • 100 uM (2.29923, 2.20247)
  • 200 uM (2.11511, 2.12708)
  • 300 uM (0.816889, 0.862665)
  • 400 uM (1.17488, 1.15506)
  • 500 uM (0.8447, 0.874413)
  • R2 (-0.5944, -0.5973)


8/14, 2008 Trial #3 (by 佳妤)

  • 50, 100, 200, 300, 400, 500, 1000, 10000 uM pi
  • dilution process: from 10 mM stock to 1000 uM, 1000 uM to 500 uM, 400 uM, 300 uM, 200 uM, 100 uM and 50 uM
  • test on Thermo Scientific Varioskan Flash and BECKMAN DU-640
  • reaction time 10 mins

NYMU IGEM08 pi standard curve 5N H2SO4 20080814 trial 32.png

  • measure (Thermo Scientific Varioskan Flash, BECKMAN DU-640)
  • blank (0.181186, n/a)
  • 50 uM (0.895658, 0.8621)
  • 100 uM (1.09361, 1.3422)
  • 200 uM (1.15711, 1.4054)
  • 300 uM (0.746172, 1.1240)
  • 400 uM (0.766937, 0.9599)
  • 500 uM (0.676912, 0.7468)
  • 1000 uM (0.669627, 0.6087)
  • 10000 uM (1.38565, 1.5171)
  • R2 (0.6683, 0.4931)


8/14, 2008 Trial #3 (by 志賢)

  • 50, 100, 200, 300, 400, 500, 1000, 10000 uM pi
  • dilution process: from 10 mM stock to 1000 uM, 500 uM, 400 uM, 300 uM, 200 uM, 100 uM and 50 uM
  • test on Thermo Scientific Varioskan Flash and BECKMAN DU-640
  • reaction time 10 mins

NYMU IGEM08 pi standard curve 5N H2SO4 20080814 trial 33.png

  • measure (Thermo Scientific Varioskan Flash, BECKMAN DU-640)
  • blank (0.302876, n/a)
  • 50 uM (0.918242, 0.8443)
  • 100 uM (0.942614, 0.9828)
  • 200 uM (0.960502, 1.1096)
  • 300 uM (0.747011, 0.8210)
  • 400 uM (0.653061, 0.6067)
  • 500 uM (0.542805, 0.4055)
  • 1000 uM (0.637159, 0.5706)
  • 10000 uM (1.35177, 1.5015)
  • R2 (0.7528, 0.7101)


8/14, 2008 Trial #3 (by Jesse)

  • 50, 100, 200, 300, 400, 500, 1000, 10000 uM pi
  • dilution process: from 10 mM stock to 1000 uM, 500 uM, 400 uM, 300 uM, 200 uM, 100 uM and 50 uM
  • test on Thermo Scientific Varioskan Flash and BECKMAN DU-640
  • reaction time 10 mins

NYMU IGEM08 pi standard curve 5N H2SO4 20080814 trial 3 by Jesse.png

  • measure (Thermo Scientific Varioskan Flash, BECKMAN DU-640)
  • blank (0.26963, n/a)
  • 50 uM (0.952509, n/a)
  • 100 uM (1.20558, n/a)
  • 200 uM (0.964501, n/a)
  • 300 uM (0.709601, n/a)
  • 400 uM (0.620946, n/a)
  • 500 uM (0.527095, n/a)
  • 1000 uM (0.429059, n/a)
  • 1000 uM (0.626158) 30 sec. delay measure
  • 10000 uM (1.38782, n/a)
  • R2 (0.5871, n/a)


8/14, 2008 Trial #3 Remark

  • all trials show when phosphate above 300 uM to 1000 uM, the measures are not linear as expectation
  • the reaction time 10 mins may not be the best choice when we don't know anything about the kinetics
  • two instruments (Thermo Scientific Varioskan Flash and BECKMAN DU-640) do not have significant measuring difference


8/21, 2008

  • 500, 1000, 10000 uM pi
  • dilution process: from 10 mM stock to 1000 uM, 1000 uM to 500 uM
  • test on BECKMAN DU-640 to perform full spectrum scanning: 200 nm is the responsive spectrum

NYMU IGEM08 pi standard curve 200nm 20080821 by blent.png

  • 500 uM (0.0233)
  • 1000 uM (0.0338)
  • 10000 uM (0.1375)
  • R2 (0.9993)


8/21, 2008

  • 100, 200, 300, 400, 500, 1000, 2000, 3000, 10000 uM pi
  • dilution process: from 10 mM stock to 3000, 2000, 1000 uM; 1000 uM to 500, 400, 300, 200, 100 uM
  • test on BECKMAN DU-640 to perform full spectrum scanning: 200 nm is the responsive spectrum

NYMU IGEM08 pi standard curve 200nm 20080822 by blent.png

  • 100 uM (-0.1794)
  • 200 uM (-0.1994)
  • 300 uM (-0.1981)
  • 400 uM (-0.1919)
  • 500 uM (-0.1908)
  • 1000 uM (-0.1781)
  • 2000 uM (-0.1727)
  • 3000 uM (-0.1597)
  • 10000 uM (-0.0708)
  • R2 (0.9866)
  • remark: This method fails in MOPS medium

8/27, 2008

  • calibrated by Thermo Scientific Varioskan Flash in ddH2O

NYMU Pi measure dynamics 100 300 500 uM.png

  • 100uM, 300uM and 500uM

NYMU 20080827 pi measure dynamics time point selection.png

  • time point selection

Standard Curve in MOPS

9/3, 2008
  • calibrated by Thermo Scientific Varioskan Flash in MOPS medium

NYMU Pi measure dynamics in MOPS 100 300 500 1000 2000 3000uM.png

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