3.A. Calibrating the Quantus™ Fluorometer for Use With the QuantiFluor
®
Dye
Systems (continued)
4. Prepare the blank sample for the QuantiFluor
®
ONE dsDNA System by
adding 200μl of QuantiFluor
®
ONE dsDNA Dye to a 0.5ml PCR tube.
Prepare the blank sample for all other QuantiFluor
®
Systems by adding
200μl of QuantiFluor
®
Dye working solution prepared in Step 2 to a 0.5ml
PCR tube.
5. For the QuantiFluor
®
ONE dsDNA System, add 1µl of the standard to
200μl of QuantiFluor
®
ONE dsDNA Dye.
For all other QuantiFluor
®
Systems, add standard prepared in Step 3 to
200µl of QuantiFluor
®
Dye working solution prepared in Step 2.
6. Mix three times by pipetting slowly. When using aerosol-resistant pipette
tips, do not allow the pipette tip filter to get wet. Alternatively, vortex
tubes at a high setting for 10 seconds.
7.
Optional:
Centrifuge tubes at 2,000 ×
g
for 5–10 seconds to collect liquid at
the bottom of the tube and remove any bubbles present.
8. Incubate tubes at room temperature for 5 minutes, protected from light.
Calibration Protocol
1. Select the desired QuantiFluor
®
Dye assay from the Protocol screen on the
instrument. If this is the first time the protocol has been selected, the
Calibration screen will automatically appear. Otherwise, after selecting the
desired protocol, navigate to the Calibration screen.
2. Place the blank sample into the tube holder, and close the lid. Select “Read
Blank”, and the fluorescence in relative fluorescence units (RFU) for the
blank sample will be displayed on the screen.
3. Place the standard sample into the tube holder, and close the lid. Select
“Read Std”, and the fluorescence in RFU for the standard sample will be
displayed on the screen.
Note:
Once both the blank and standard samples have been measured, the
standard-to-blank ratio is automatically calculated as a quality check to
ensure the QuantiFluor
®
Dye working solution and blank and standard
samples were prepared properly. If the standard-to-blank ratio is lower
than the minimal accepted ratio, the screen will display “Invalid”, and the
instrument will not quantitate the unknown samples until the blank and
standard samples are prepared properly. The minimum standard-to-blank
ratio is:
• 50 for high-concentration standard curve or single standard curve protocols
• 4 for low-concentration standard curve protocols
• 3 for user-defined protocols
These ratios have been predetermined as a quality check to help ensure a
successful measurement.
Promega Corporation
·
2800 Woods Hollow Road
·
Madison, WI 53711-5399 USA
Toll Free in USA 800-356-9526
·
Phone 608-274-4330
·
Fax 608-277-2516
·
www.promega.com
Part# TM396
Printed in USA.
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