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

Standard Curve in ddH2O

 * iGEM08 How to measure phosphate

7/23, 2008 PM 4:04

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

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

7/30, 2008 PM 9:48

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

7/30, 2008 PM 10:00

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

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

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


 * reaction time 20 mins

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

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

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

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

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


 * remark: This method fails in MOPS medium

8/27, 2008

 * calibrated by Thermo Scientific Varioskan Flash in ddH2O

9/3, 2008

 * calibrated by Thermo Scientific Varioskan Flash in MOPS medium