Molecular Devices SpectraMax Paradigm User Manual page 55

Background Correction
Although background is significantly lower than with fluorescence intensity measurements,
Molecular Devices recommends that you use blanks or assay controls for background
correction. The background can be effectively measured using blank replicates. When
reading a sample with small signal, an interference can occur from the afterglow of a very
strong emitting adjacent sample that was measured just before. Such cross talk can occur
through the wall of a white 384-well plate. To prevent such interference, you can select the
Interlaced Reading option in the SoftMax Pro Software Settings dialog. This option reads
only every other well in a checkerboard pattern, and then reads the microplate again to read
the previously omitted wells.
A blank well contains everything used with the sample wells except the label and sample-
specific compounds. Do not use an empty well for a blank.
For optimum results, Molecular Devices recommends that you run replicates for all blanks,
controls, and samples. In this case, the blank value that can be subtracted is the average
value of all blanks.
Detection Limit
The detection limit is the smallest sample concentration that can be measured reliably above
the blank. Determining the detection limit requires taking a number of blank measurements
and calculating an average value and standard deviation for the blanks. The detection
threshold is defined as the average blank plus three standard deviations. If the average
sample value measures above the threshold, the sample can be detected at a statistically
significant level.
The detection limit can be described by the following equation:
Det Limit =
where conc
blank replicates, and blank and Std are average values of the replicates for the blank and
standard wells.
5014038 E
3 Stdev
blank
std – blank
conc
std
is the concentration of the standard, StDev
std
Chapter 2: Read Modes and Read Types
is the standard deviation of the
Blank
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