Biotek Synergy HT, Operator'S Manual

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Absorbance Liquid Tests
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87
Synergy HT Operator's Manual
Procedure
™
In this procedure, the prepared stock buffer solution B is referred to as
the
100% Test Solution .
1.
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.
2. 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.
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
4. Using Gen5™ or KC4™, read the microplate five times using Normal mode,
single wavelength at 340 nm, no blanking. Save the data after each read.
5. Print out the five sets of raw data, or export them to an Excel spreadsheet.
Data Reduction for Repeatability:
6. For each well, calculate the mean and standard deviation of the five
readings.
7. For each mean calculated in step 6, calculate the allowed deviation using
the repeatability specification for a 96-well plate of ±1.0% ± 0.005 OD
(Mean x 0.010 ± 0.005).
8. For each well, compare the standard deviation calculated in step 6 with the
allowed deviation calculated in step 7. The standard deviation should be
less than the allowed deviation.
Example: Five readings in well A1 of 0.802, 0.802, 0.799, 0.798, and 0.801
will 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 the
0.005 (0.008 + 0.005) equals
0.013 , which 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.
• 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