Molecular Devices SpectraMax GEMINI EM, Operator'S Manual

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Chapter 3
3-4 SpectraMax GEMINI EM Operator’s Manual
When reading is complete, the drawer of the instrument will open, allowing
you to remove the microplate. If the incubator is on, the drawer will close
again after approximately 10 seconds. If you return to the SpectraMax GEM-
INI EM and
fi nd the drawer closed after a reading has fi nished, press the
[Drawer]
key. When the drawer opens, you may remove the microplate.
Operation
Overview
The following steps provide a quick reminder of the basic operating proce-
dures required to perform an assay using the SpectraMax GEMINI EM.
1) Turn on the power switch of the SpectraMax GEMINI EM (located on the
back panel). The microplate drawer will open automatically.
2) If you wish to regulate the temperature inside the microplate chamber,
touch the
[Temp on/off]
(incubator) key to turn the incubator on and
bring the microplate chamber to the default temperature of 37.0°C. The
microplate drawer will close.
3) If the incubator is on, the LCD will show the current temperature along
with the temperature set point. To change the set point (to any setting
from a4° to 45°C), press the up or down arrow keys.
4) Select the desired instrument settings (read mode, type of analysis, tem-
plate, etc.) using SoftMax Pro software on the external computer.
5) If you are performing kinetic analysis, add substrate at this time.
6) Load the prepared microplate into the drawer, being sure to match well
A1 with the A1 mark on upper left-hand corner of the drawer.
7) Using SoftMax Pro, start the reading.
Optimizing
Assays
Introduction
The optimum instrument settings for detection of a particular fl uorophore
depend on a number of different factors. Settings that can be adjusted for
assay optimization include the excitation and emission wavelengths, emis-
sion cutoff
fi lter, readings per well, the PMT voltage, and the temperature of
the reading chamber.
• The excitation and emission wavelengths may be set in 1-nm increments
within the range of the instrument (250-850 nm). A procedure to optimize
excitation and emission wavelengths for a given assay is outlined in the
next section.
• The 15 emission cutoff fi lters assist in reducing background. Sources of
background include stray excitation light and native fl uorescence of plate
materials, sample constituents, and solvents (including water). The default
setting allows the instrument and SoftMax Pro software to determine which
cutoff
fi lter should be used (see Table 3.1 for default settings) in endpoint
and kinetic modes. The spectral scan mode default uses no cutoff fi lter.
• The number of readings per well may vary between 1 (used for a quick esti-
mate) and 30 (for very precise measurements). The default number of read-
ings per well varies with the read mode: for
fl uorescence, the default is 6,
and for luminescence, the default is 30.
• The voltage of the photomultiplier tube may be set to low (for higher con-