Bio-Rad Fluorescent Assay Kits - Manual (Page 4)

Section 4
Experimental Procedures
The FluorAce ß-galactosidase Reporter Assay Kit has been designed
for use with any fluorometer system. The following section describes the
procedure to be used with the Bio-Rad VersaFluor™ Fluorometer. If
another instrument is used, refer to the instruction manual regarding spe-
cific operation details.
4.1 Fluorometer Set-up
1. Turn on the fluorometer to allow instrument to warm up for at least
20 minutes. If the instrument is not fully warmed up, the reading may
be irreproducible.
2. Insert a 360 nm excitation filter and a 460 nm emission filter into the
fluorometer.
3. Set the gain to LOW and adjust the range to read 00000.
4.2 Sample Measurement Guidelines
Follow the guidelines below to insure accurate results.
• Warm up the instrument for at least 20 minutes
• Always use calibrated pipets to insure accurate pipetting
• Mix standards and sample completely by using a disposable
transfer pipet
• Remove any air bubbles in the cuvette
• Hold fluorometer cuvettes by the upper edges; avoid handling the
four optically-clear sides
• Clean the cuvette sides with a lint-free tissue
• Read all standards and samples at ambient temperature
• While making a reading, keep the samples in the fluorometer
only. This helps reduce photobleaching.
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Component Concentration Volume
1x Reaction Buffer (green cap) 1x 100 ml
4-methylumbelliferyl ß-D-galactopyranoside (MUG) 100 mM 600 µl
10x Stop Buffer (red cap) 10x 50 ml
4-methylumbelliferone (4MU) 1.0 mM 400 µl
ß-galactosidase 24–38,000 Units* 100 µl
* One unit of ß-galactosidase hydrolyzes 1 nanomole of 4-methylumbelliferyl ß-D-
galactoside per minute at pH 7.5 at 37 °C.
2.2 Materials Required
ß-mercaptoethanol (catalog number 161-0710).
2.3 Storage and Stability
All components are guaranteed for 12 months from the date of pur-
chase when used as described in this manual and stored as described in
the table below:
Component Storage Temperature
1x reaction buffer Room temperature
Stop buffer (10x or 1x) Room temperature
4-methylumbelliferyl ß-D-galactopyranoside (MUG) 4 °C
4-methylumbelliferone (4MU) 4 °C
ß-galactosidase 4 °C
Section 3
Preparation of Stock Solutions
1x Stop Buffer
Prepare 1x Stop Buffer by mixing 50 ml of 10x Stop Buffer with 450 ml
of distilled water. Store solution at room temperature.
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