Liquid Testing | 41
3. Carefully 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
Read the Plate
1. Using Gen5, read the microplate five times using Normal mode, single
wavelength at 340 nm, no blanking. Save the data after each read.
2. Print out the five sets of raw data, or export them to an Excel spreadsheet.
Analyze the Results
1. The plate is read five times at 340 nm. Calculate the Mean OD and Standard
Deviation of those five reads for each well in columns 1–6.
2. For each well in columns 1–6, calculate the allowed deviation using the
repeatability specification for a 96-well plate: ± 1% ± 0.005 OD (Mean x
0.010 + 0.005). For each well, its standard deviation should be less than its
allowed deviation.
Example: Five readings in well A1 of 0.802, 0.802, 0.799, 0.798, and 0.801
result in a mean of 0.8004 and a standard deviation of 0.0018. The mean
multiplied by 1.0% (0.8004 * 0.010) equals 0.008, and when added to 0.005
equals 0.013; this is the allowed deviation for well A1. Since the standard
deviation for well A1 is less than 0.013, the well meets the test criteria.
3. Calculate the results for Linearity:
• For each of the three Test Solutions, calculate the average Mean OD for
the wells containing that solution (mean of wells A1 to H2, A3 to H4,
and A5 to H6).
• Perform a regression analysis on the data to determine if there is
adequate linearity. The three average Mean OD values are the “Y”
values. The solution concentrations are the “X” values (1.00, 0.75, 0.50).
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.
Epoch Operator’s Manual
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