University of Texas at San Antonio
SOP No.: 3 version 1.0
Effective Date: 9/19/2008
Page 1 of 3
Title: Luciferase Assay
Approved:
Karl Klose
UTSA PI
Author: Jeff Barker
Date: 9/19/2008
1. INTRODUCTION
The luciferase assay is a useful technique that allows the direct quantification of
firefly or bacterial luciferase activity and has many applications in molecular biology.
2. PURPOSE
This protocol describes the technique of quantifying the luciferase (or light) activity
from a bacteria expressing luciferase. Luciferase has been used for screening
promoter activity as well as detecting the overall survival of an organism in a host or
tissue culture cell. Samples expressing firefly luciferase require luciferase reagent
(luciferin, Promega) whereas bacterial luciferase requires no extra reagents for the
assay.
3. RESPONSIBILITY
Research Technologist: performs the SOP
Technical Specialist: reviews the data, calculations and interpretations
Principal Investigator: reviews the data and interpretations
4. SCOPE
This procedure is used by the UTSA team under the direction of Dr. Karl Klose
5. PRECAUTIONS
Sterility and temperature maintenance are critical steps in successfully preparing the
bacterial samples for the luciferase assay. Be sure to use sterile media and tubes
during the sample preparation procedure. It is important to make buffers and reagents
fresh, ~30 minutes prior to experimental procedures.
6. MATERIALS AND EQUIPMENT
6.1 Luminometer (Lumat LB 9507, EG&G Berthold)
6.2 Sonicator (VirSonic Ultrasonic Cell Disrupter)
6.3 Luciferase reagent (Promega, E1500)
6.4 Luciferase buffer (See Procedure Section 7.1.1)
6.5 Nichipet EX Plus pipettes, calibrated annually (p10, p20, p200, p1000)
6.6 Glass tubes (provided with luminometer)
University of Texas at San Antonio
SOP No.: 3 version 1.0
Effective Date: 9/19/2008
Page 2 of 3
Title: Luciferase Assay
Approved:
Author: Jeff Barker
Karl Klose
UTSA PI
Date: 9/19/2008
7. PROCEDURE
7.1 Reagent Preparation
7.1.1 1X Luciferase Buffer (25mM Tris, 2mM DTT, 2mM EDTA, 10% glycerol,
1% TritonX-100)
1.25 ml of 1 M Tris
100 ul of 1 M DTT
200 ul of 0.5 M EDTA
5 ml of 10% Glycerol (or 6.25 ml 80%)
0.5 ml 100% TritonX-100
Bring up to 50ml with H2O, store at 4ºC in 50 ml Conical Tube
7.2 Preparation of samples
7.2.1 Grow up 1 ml of Bacteria culture overnight from a colony on a plate, use
LB to grow E. coli and TSB for Francisella cultures
7.2.2 Take 100 ul of overnight and put into 900 ul of fresh media and let grow 3
hours to log phase
7.2.3 Take 100 ul from log phase (OD600 of ~0.8) culture and put into 900 ul of
1X luciferase buffer
7.2.4 Sonicate for 15 seconds (Samples can be frozen at -20ºC at this point or
assayed immediately)
7.2.5 Take out luciferase reagent from -80C freezer to thaw at room temperature
and use immediately.
7.2.6 Assay 10 ul from sonicated cells in luminometer
7.3 Luminometer Operation (procedure will vary depending on equipment
model)
7.3.1 Turn on machine
7.3.2 Unload water from reservoir
7.3.3 Replace water bottle with substrate bottle (Promega luciferin)
7.3.4 Prime machine with substrate
7.3.5 Measure luciferase activity by adding glass tube containing sample to the
luminometer. Acceptable read out of sample is any reading that is
significantly over your negative control. In general, anything over 100 is
considered positive. Units will be given as RLU (reactive light units).
7.3.6 Unload substrate
University of Texas at San Antonio
SOP No.: 3 version 1.0
Effective Date: 9/19/2008
Page 3 of 3
Title: Luciferase Assay
Approved:
Author: Jeff Barker
Karl Klose
UTSA PI
7.3.7
Date: 9/19/2008
Switch Substrate for water and wash the line 2x with water to clear the
tubing of substrate
8. QUALITY CONTROL
Temperatures of incubators are noted with each usage
Luminometer is calibrated and serviced annually (this is quality assurance)
Pipettes are calibrated annually (this is quality assurance)
9. REFERENCES
None
10. CALCULATIONS AND FORMULAS
RLU/ml/ODU are calculated thus: The value obtained from the luminometer (reactive
light units, RLU) is divided by the volume (ml) of culture added to the luminometer
tube, divided by the OD (optical density) of the bacterial culture. Thus, volumes of
culture (ml) added and growth phase (OD) of the culture are incorporated into the
RLU (luciferase) data.
11. GLOSSARY
 ml- milliliter
 ul- micro liter
 cm- centimeter
 C- centigrade
 LB- Luria broth
 TSB- Tryptic Soy Broth
12. APPENDICES
None