Biotek Synergy HTX, Operator'S Manual, Page: 135 / 184

Absorbance Liquid Tests |
113
Prepare the Plate
•
Pipette 200 µL of the concentrated solution from Tube 1 into each well of the
first column, A1 to H1, of a new flat-bottom microplate.
• Pipette 200 µL from each of the remaining tubes into the wells of the
corresponding column of the microplate (Tube 2 into wells A2 to H2, Tube 3
into wells A3 to H3, and so on).
Linearity and Repeatability Tests
1.
Using Gen5, read the microplate prepared above
five times
using Normal
mode, dual wavelength at 450/630 nm. Save the data after each read.

Do not discard the plate; you will use it for the Alignment test.
2.
Print out the five sets of
Delta OD
data, or export them to an Excel
spreadsheet.
3.
Calculate the results for Linearity:
•
Calculate the mean absorbance for each well, and average the means for
each concentration.
•
Perform a regression analysis on the data to determine if there is
adequate linearity.
Since it is somewhat difficult to achieve high pipetting accuracy when
conducting linear dilutions, an R Square value of at least 0.99 is considered
adequate.
4.
Calculate the results for Repeatability:
• Calculate the mean and standard deviation for the five readings taken in
Step 1 at each concentration. Only one row of data needs to be analyzed.
• For each mean below 2.000 OD, calculate the allowed deviation using
the repeatability specification for a 96-well plate of ± 1.0% ± 0.005 OD. If
above 2.000 OD, apply the ± 3.0% ± 0.005 specification.
• The standard deviation for each set of readings should be less than the
allowed deviation.
Example: Absorbance readings of 1.950, 1.948, 1.955, 1.952, and 1.950 will result
in a mean of 1.951, and a standard deviation of 0.0026. The mean (1.951)
multiplied by 1.0% (1.951 x 0.010) = 0.0195, which, when added to the 0.005
(0.0195 + 0.005) = 0.0245 OD, which is the allowable deviation. Since the
standard deviation is less than this value, the reader meets the test criteria.
Repeatability Specification:
± 1.0% ± 0.005 OD from 0.000 to 2.000 OD
± 3.0% ± 0.005 OD from 2.000 OD to 3.000 OD
Synergy HTX Operator's Manual