38 | Chapter 4: Instrument Qualification
• 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 & 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. For example, using Microsoft Excel:
¾ In a spreadsheet, create two columns labeled “X” and “Y”. As
displayed in the table
earlier, enter the actual absorbance values in
column X, and enter the
expected absorbance values in column Y.
¾ Select
Tools > Data Analysis > Regression. Identify column X
as the “Input X Range” and column Y as the “Input Y Range.”
¾ Click
OK to perform the analysis, the results of which will be
output in a separate sheet.
If the Data Analysis command is not available on the Tools menu, you
may need to install the Analysis ToolPak in Excel. Consult Excel’s help
system for assistance.
Because 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.
BioTek
®
Instruments, Inc.
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