Molecular Devices SpectraMax Plus 384 User Manual page 22

VersaMax, SpectraMax 340PC384, 190, Plus 384 Microplate Readers User Guide
Place a standard 1 cm cuvette that contains the aqueous/solvent mixture you use for the
plate samples into the cuvette port. The cuvette must be in place when you read the plate.
When you click Read, the instrument first makes the 900 nm and 1000 nm measurements in
the cuvette and then makes the designated measurements in the plate. The software
temporarily stores the cuvette values and uses the cuvette values in the PathCheck
calculations for the plate samples.
The Cuvette Reference data does not display in a Cuvette Set section. The software uses the
Cuvette Reference data for PathCheck Pathlength Measurement Technology calculations on
the data the instrument collects from the plate. You can use the accessors in the Formula
Editor dialog to obtain these values. See the !PathCheckLm1000 and !PathCheckLm900
accessor in the SoftMax Pro Data Acquisition and Analysis Software Formula Reference
Guide.
Eliminating Pathlength Independent Component
Raw OD measurements of plate samples include both pathlength-dependent components
(sample and solvent) and a pathlength-independent component (OD of plate 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.
Use a Plate Blank
You can use this method if all samples in the plate are the same volume and the read does
not depend on the PathCheck technology to correct for variability in volumes.
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 the
samples. Do not use an empty well for a blank.
The instrument automatically subtracts the average of the blank wells from each of the
samples. The OD of the plate material is subtracted as part of the blank.
3. Select the Use Plate Blank check box in the Data Reduction dialog.
Use a Plate Background OD
If your sample volumes are not identical or if you choose not to use a Plate Blank, then you
must use a Plate Background OD. Omitting a Plate Background OD results in artificially high
values after being normalized by the PathCheck technology.
To determine the Plate Background OD:
1. Fill a clean plate with water.
2. Read at the wavelengths you will use for the samples.
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Note: After you read a plate with PathCheck technology turned on, the software
stores PathCheck information permanently in the document. You can apply or not
apply PathCheck technology to the absorbance values. If you do select to use
PathCheck technology for the plate read, you cannot apply the PathCheck Pathlength
Measurement Technology feature after the read.
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