1.3
1.3 kdorfman Mon, 08/19/2013 - 17:18Corrections
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 |