User-Defined Protocol For Other Dye Chemistries; Calibrating The Quantus™ Fluorometer For Use With Other Dye Chemistries - Promega Quantus Fluorometer Operating Manual

4. Save the calibration data by selecting "Save". Proceed to Section 3.D.
3.B. User-Defined Protocol for Other Dye Chemistries
Fluorescent dyes other than QuantiFluor
Quantus™ Fluorometer, provided they have the proper excitation and emission
wavelengths (see Section 1.C). To quantify samples using other dyes, up to
three user-defined protocols may be created. User-defined protocols require
calibration (Section 3.C).
1. From the Protocol screen, select "New" from the menu list.
2. Enter the name of the protocol by using the up or down buttons. When the
desired character is displayed, press the right button to select it, and move
to the next box on the screen.
3. Enter the high-concentration standard value by choosing 1–1,000. Select the
concentration units and sample volume.
Note: The standard value is entered when setting up the user-defined
protocol. After calibration, you can change the units and volume from the
Main screen. Several factors will influence the standard value entered,
including the concentration units, mass of nucleic acid per tube and sample
volume. The instrument firmware assumes a 200µl final volume. For
example, with the concentration units set to ng/µl, the sample volume set
to 1µl, and the mass of nucleic acid at 100ng per tube, the standard value
entered is 100/200 = 0.5.
4. Choose the appropriate fluorescence channel:
Red: Excitation 640nm shortpass, emission 660–720nm
Blue: Excitation 495nm shortpass, emission 510–580nm
5. Select "Save".
Note: To edit or delete a user-defined protocol, select "Edit" next to the
protocol name.
3.C. Calibrating the Quantus™ Fluorometer for Use With Other Dye Chemistries
User-defined protocols must be calibrated using blank and standard samples
prepared similarly to those described in Section 3.A. You may choose another
standard, but the instrument assumes the total assay volume in the tube is
200µl. If less than 200µl is used, the sample may not be completely within the
light path, causing inaccurate sample readings, and the concentration
calculation must be corrected, since the instrument calculates based on a 200µl
assay volume.
1. Prepare the blank and standard samples as directed in Section 3.A.
Alternatively, you may use a different standard value in the user-defined
protocol. We recommend a minimum assay volume of 200µl. Note that the
instrument software assumes a linear relationship when calculating results
from a single-point setup (blank and standard). Be sure to verify, through a
dilution series, that any dye dilution or standard combination is accurate.
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Revised 1/20
® Systems may be used with the
Part# TM396
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