Optimizing Fluorescence Assays; Optimizing Absorbance Assays; Excitation And Emission Wavelengths - Molecular Devices SpectraMax M3 User Manual

Operation

Optimizing Fluorescence Assays

Optimizing Absorbance Assays

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3. Select the Control > Read command or press the Read button in
SoftMax Pro Software to start the plate read.
4. When reading is complete, the drawer of the instrument opens,
allowing you to remove the microplate. If the incubator is on,
the drawer closes again after approximately 10 seconds.
5. If you return to the SpectraMax instrument and find the drawer
closed after a reading has finished, press the
When the drawer opens, you can remove the microplate.
For more information about configuring the software for plate reading,
please consult the SoftMax
The wavelength of the transmitted light can be adjusted in 1-nm
increments between 200 nm and 1000 nm. Guke also allows reading up
to four wavelengths per plate. This enables reference wavelength
readings such as A260 and A280 for nucleic determination.
An appropriate plate blank should be applied. Unless the user suspects
that there is significant well-to-well variability due to the thickness and
optical properties of the plate, the use of Pre-Read Plate in the SoftMax
Pro program is not required. Instead, we recommend using appropriate
plate blanks or group blanks in the Template dialog box of the Plate
section in the SoftMax Pro program. For discussion of the different
types of blanking, please refer to the SoftMax
Guide.
If desired, the PathCheck Technology feature in SoftMax Pro program
can be activated to normalize the data to a 1-cm pathlength.
The optimum instrument settings for detection of a particular
fluorophore depend on a number of different factors. Settings that can
be adjusted for assay optimization include the excitation and emission
wavelengths, emission cutoff filter, readings per well, the PMT voltage,
and the temperature of the reading chamber.
Another important factor that is independent of the instrument but
which affect assays optimization is the Stokes' shift. When the Stokes'
shift is very small, optimizing the excitation and emission wavelengths
and correct cutoff filter choices are very important.

Excitation and Emission Wavelengths

The excitation and emission wavelengths may be set in 1-nm
increments between 250 nm and 850 nm. A procedure to optimize
excitation and emission wavelengths for a given assay is outlined
below.
Pro Software User Guide.
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key.
DRAWER
Pro Software User
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0112-0115 F