36 | Chapter 4: Instrument Qualification
After pipetting the diluted test solution into the microplate,
we strongly recommend waiting 20 minutes before reading
the plate to allow any air bubbles in the solution to settle and
the meniscus to stabilize.
4. Read the microplate five times at 405 nm using normal reading mode,
single wavelength, no blanking (“Normal” plate position). Save the data
after each read (“Normal” plate position).
5. Rotate the microplate 180° so that well A1 is now in the H12 position. Read
the plate five more times (“Turnaround” plate position) saving the data
after each read.
The ten sets of raw plate data can be exported to an Excel spreadsheet
using Gen5. The mathematical computations described below may then be
performed and the template kept for future data reduction.
Calculations:
6.
Calculate the mean value for each physical well location in columns 1 and 2
for the five plates read in the Normal position, and then again for the five
plates read in the Turnaround position. This will result in 32 mean values.
7. Perform a mathematical comparison of the mean values for each microwell
in its Normal and Turnaround positions (A1/H12, A2/H11, B1/G12,
B2/G11, and so on). In order to pass this test, the differences in the
compared mean values must be within the accuracy specification for the
instrument.
For example:
If the mean value for well A1 in the Normal position is 1.902, where the
specified accuracy is ± 1% ± 0.010 OD, then the expected range for the mean
of the same well in its Turnaround (H12) position is 1.873 to 1.931 OD.
1.902 * 0.01 + 0.010 = 0.029; 1.902 – 0.029 =
1.873; 1.902 + 0.029 = 1.931
Accuracy Specification:
For comparison in this test, the following accuracy specifications are
applied, using Normal reading mode and a 96-well microplate.
± 1% ± 0.010 OD from 0.000 OD to 2.000 OD
± 3% ± 0.010 OD from 2.000 OD to 3.000 OD
BioTek
®
Instruments, Inc.
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