Biotek EL800, Operator'S Manual

Page: 151 / 218

Liquid Testing
|
125
ELx800 Operator's Manual
Procedure
1. Check the absorbance of a sample of buffer solution A or B at 340 nm on the
microplate reader. This solution, which will be referred to as the
100%
Test Solution , will have an optical density (absorbance) of approximately
0.700 to 1.000. This value is not critical, but it should be within this
absorbance range.
If low, adjust up by adding
D
-NADH powder until the solution is at least at
the lower end of this range. Do not adjust if slightly high.
2. Carefully prepare a 75% Test Solution by diluting 15 ml of the 100% Test
Solution:
If using the Sigma PBS Solution, use 5 ml as the diluent.
If using the 10x Concentrate PBS Solution, mix one part of the
concentrate with nine parts of DI water. Use 5 ml of this solution as the
diluent.
3. Carefully prepare a 50% Test Solution by diluting 10 ml of the 100% Test
Solution:
If using the Sigma PBS Solution, use 10 ml as the diluent.
If using the 10x Concentrate PBS Solution, mix one part of the
concentrate with nine parts of DI water. Use 10 ml of this solution as the
diluent.
4. Pipette the three solutions into a new 96-well microplate:
150 µl of the 100% Test Solution into all wells of columns 1 and 2
150 µl of the 75% Test Solution into all wells of columns 3 and 4
150 µl of the 50% Test Solution into all wells of column 5 and 6
After pipetting the diluted test solution into the microplate and before
reading the plate, we strongly recommend shaking the plate for four
minutes. This will allow any air bubbles in the solution to settle and the
meniscus to stabilize. If a plate shaker is not available, wait 20
minutes after pipetting the diluted test solution before reading the
plate.
5. Read the microplate five times using Normal Mode, single wavelength at
340 nm, no blanking (or blank on air).
6. Print the five sets of raw data or export it to an Excel spreadsheet using
Gen5™, KC4™, or KCjunior™.
If you are exporting the data to an Excel spreadsheet, perform the
calculations described on the following page, and keep the spreadsheet
for future tests.