PowerWave Operator’s Manual 4-17
5) Perform a percentage dilution, beginning with 100% of the original dense
solution in the first tube, 90% of the original solution in the second tube;
80% in the third tube, all the way to 10% in the last tube.
Dilute using amounts of the remaining 0.5% solution of distilled water
and Tween 20, as shown in Table 4-2.
6. Remove a 200 ml aliquot from each tube, and dispense it into the
appropriate microplate well. There should be enough solution to have
several replicates, if desired.
7. Read the microplate at 405 nm to determine the absorbance, and then
graph the results, plotting absorbance versus percentage. You should get
a straight line passing through zero, if your pipetting is accurate.
8. The choice of dilutions is strictly arbitrary, as is the absorbance of the
original solution. Use Table 4-2 as a model for calculating the expected
absorbances of a series of dilutions, given a different absorbance of the
original solution.
Verification of Alignment
• Use a new microplate, free from dust and scratches which could
affect the readings.
• Fill the microplate with the time-stable solution you want to
measure, such as the solution suggested for the Linearity Test. Use a
filter wavelength corresponding to the maximum absorbance of
your solution.
• Use different solution volumes (not less than 100 microliters) and
densities. For example, put 100 microliters in rows A through D,
and 150 microliters in rows E through H.
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