1.3

Corrections

Read the plate only once

Pipet 150 µL of each unknown in E3 - 12 as needed

Programs

  • Serial2&5_Abs
  • Serial2&5_FL
  • E1 &E2: blank E3-E12 for unknowns
  • F1 - F12: dilute by 2
  • G1 - G12: dilute by 5

Materials:

  • ~1 mL 1mM fluorescein (try to make it right via Abs)
    • E = 50358/M
    • A = 50358/M * C
    • at 10 µM, A = 0.5038
    • dilute 1 mM 1/100 to make 10 µM, take Abs
  • 5 mL 10 mM NaOH
  • black plate from last time (or new one if old one has no clean wells)

Conversion factors

Abs = 59.7*(mM)-0.341 up to mM=0.03125

FL = 10^6 * mM +173.06 up to mM = 0.008

FL = 2 X 10^6 * mM + 19.624 up to mM = 0.00195

Unknowns

  • Make dilution series from the "1mM" stock.
  • find lowest useful concentration for Abs. Dilute slightly for unknown 1 - must be done with FL. Abs is bad below 0.0005 and meaningless below 5 x 10^-7mM
  • find a concentration too high for fluorescence, but OK for abs, e.g., 0.05.
  • find a concentration too high for either. must be >0.125

2013:

label ~mM Abs FL
P 0.0001 ~0 210
Q 0.015 0.8699 16496
R 0.1 4.955 26780
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test-fluorescein_ps1.xlsx50.12 KB