Molecular Devices SpectraMax Paradigm User Manual page 26

SpectraMax Paradigm Multi-Mode Detection Platform User Guide
Eliminating the Pathlength-Independent Component
Raw OD measurements of microplate samples include both pathlength-dependent
components (sample and solvent) and a pathlength-independent component (OD of
microplate material). The pathlength-independent component must be eliminated from the
calculation to get valid results that have been normalized by the PathCheck technology. You
can do this using a plate blank or using a plate background constant.
Using a Plate Blank
This method can be used if all samples in the microplate are the same volume and you are
not depending on the PathCheck technology to correct for variability in volumes.
To use this method:
1. Designate a minimum of one well (preferably several) as Plate Blank.
2. Pipette buffer (for example, your sample matrix) into those wells and read along with
your samples. Do not use an empty well for a blank.
The SoftMax Pro Software automatically subtracts the average of the blank wells from
each of the samples. The OD of the microplate material is subtracted as part of the blank.
3. Make sure that Use Plate Blank is checked under Other Options in the Data Reduction
dialog.
Using a Plate Background Constant
If your sample volumes are not identical or if you choose not to use a Plate Blank, then you
must use a Plate Background Constant. Omitting a Plate Background Constant results in
artificially high values after being normalized by the PathCheck technology.
To determine the Plate Background Constant:
1. Fill a clean microplate with water.
2. Read at the wavelengths that you will be reading your samples.
The average OD value is the Plate Background Constant. If you intend to read your samples
at more than one wavelength, there should be a corresponding number of Plate Background
Constant values for each wavelength.
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Note: It is important that you put water in the wells and not read a dry microplate for
the Plate Background Constant. A dry microplate has a slightly higher OD value than a
water-filled microplate because of differences in refractive indices. Using a dry
microplate results in PathCheck technology normalized values that are lower than
1 cm cuvette values.
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