Jenny’s Creb Response Element (CRE)-Luciferase Assay
Experimental Design
Plate 1: Transfected with CRE-Lucif and Renilla
A
B
A
B
A
B
C
C
C
D
D
D
Plate 2: Tranfected with CRE-Lucif and Renilla
E
F
E
F
E
F
G
G
G
H
H
H
Plate 3: Transfected with CRE-Lucif and Renilla; 3 wells transfected with Control Lucif and Renilla
I
J
Control-Lucif
(blank)
I
J
Control-Lucif
(blank)
I
J
Control-Lucif
(blank)
Protocol
Day 1 (Day Prior to Transfection):
1. Seed 100,000 MCF7 cells/well of a 12 well plate in PRF-DMEM + 10% CD-FBS + 2 mM L-glutamine
WITHOUT AB/AM. Label the plate according to the above experimental design.
Day 2 (Day of Transfection):
1. Wash cells 1X with PBS.
2. Change media to PRF-DMEM + 0.5 % CD-FBS + 2 mM L-glutamine WITHOUT AB/AM. Stick back
in the incubator during the preparation for transfection.
3. Prepare Fugene: For each well to be transfected, dilute the appropriate amount of Fugene6 Reagent into
50 ul Optimem. Optimem must be pipetted first. Add the fugene directly to the center of the tube. Do not
let it come in contact with the tube walls. Mix and incubate this Fugene6 mixture for 5 min at RT.
Mastermix 1: CRE-Lucif and Renilla
# of wells to be transfected:_____ + ____ (pipet error) = _____ “transfections”
50 ul Optimem/well * ____wells to be transfected with CRE-Lucif and Renilla= _____ ul Optimem
Using a 3:1 Optimem:Fugene ratio, and for a 12 well plate that is 48.5 ul Optimem and 1.5 ul Fugene6/well
1.5 ul Fugene6/50 ul Optimem = x ul Fugene6/ _____ ul Optimem = ___ ul Fugene6 needed
Mastermix 2: Control-Lucif and Renilla
# of wells to be transfected:_____ + ____ (pipet error) = _____ “transfections”
50 ul Optimem/well * ____wells to be transfected with CRE-Lucif and Renilla= _____ ul Optimem
Using a 3:1 optimem:fugene ratio, and for a 12 well plate that is 48.5 ul Optimem and 1.5 ul Fugene6/well
1.5 ul Fugene6/50 ul Optimem = x ul Fugene6/ _____ ul Optimem = ___ ul Fugene6 needed
4. Prepare DNA: For each well to be transfected, dilute the appropriate amount of CRE-Lucif OR ControlLucif along with Renilla construct into the Fugene 6/Optimem mixture. Incubate for ~30-45 min at RT
to allow DNA/fugene complexes to form.
Concentration of CRE-Lucif AND Control-Lucif is 500 ng/ml; Concentration of Renilla is 1 ng/ul (if none
left, prepare another stock from the primary stock of 401.6 ug/ml).
Mastermix 1:
300 ng CRE-Lucif
3 ng Renilla plasmid
300 ng CRE-Lucif / 500 ng/ml = ____ ul Creb-Lucif plasmid
# of wells to receive CRE-Lucif: ____
______ * ____ ul Creb-Lucif plasmid = _______ ul CRE-Lucif plasmid required for these wells
3 ng Renilla plasmid / 1 ng/ul = ____ ul Renilla plasmid
# of wells to receive Renilla (same as above): _____
_____ * ____ ul Renilla plasmid = _______ ul Renilla plasmid required for these wells
Mastermix 2:
300 ng Control-Lucif
3 ng Renilla plasmid
300 ng Control-Lucif / 500 ng/ml = ____ ul Creb-Lucif plasmid
# of wells to receive Control-Lucif: ____
______ * ____ ul Control-Lucif plasmid = _______ ul Control-Lucif plasmid required for these wells
3 ng Renilla plasmid / 1 ng/ul = ____ ul Renilla plasmid
# of wells to receive Renilla (same as above): _____
_____ * ____ ul Renilla plasmid = _______ ul Renilla plasmid required for these wells
5. Add 100 ul of this combined DNA/fugene/Optimem mix directly to the cells, in a dropwise fashion all
over the well. Gently rock the plate back and forth to ensure even transfection.
6. Incubate the cells overnight at 37°C / 5 % CO2.
Day 3: Day of Luciferase Assay:
1. Prepare treatment medias, thaw CMs (depending on the assay)
2. Aspirate media and replace with treatment media. Allow cells to incubate in treatment media for 30 min
at 37°C / 5 % CO2.
3. Prepare the luciferase reagents in the interim, following Promega instructions.
4. Begin luciferase assay, following Promega instructions.