Luciferase Assay Protocol

Luciferase Assay Protocol ( Click to Download )

Experimental Design

Plate 1: Transfected with CRE-Lucif and Renilla

Plate 2: Tranfected with CRE-Lucif and Renilla

Plate 3: Transfected with CRE-Lucif and Renilla; 3 wells transfected with Control Lucif and Renilla

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 µl 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 µl Optimem/well * ___wells to be transfected with CRE-Lucif and Renilla= ____ µl Optimem

Using a 3:1 Optimem:Fugene ratio, and for a 12 well plate that is 48.5 µl Optimem and 1.5 µl Fugene6/well

1.5 µl Fugene6/50 µl Optimem = x µl Fugene6/ ____ µl Optimem _= ___ µl Fugene6 needed

Mastermix 2: Control-Lucif and Renilla
# of wells to be transfected:___ + ____ (pipet error) = ___ “transfections”

50 µl Optimem/well * ___wells to be transfected with CRE-Lucif and Renilla= ____ µl Optimem

Using a 3:1 optimem:fugene ratio, and for a 12 well plate that is 48.5 µl Optimem and 1.5 µl Fugene6/well

1.5 µl Fugene6/50 µl Optimem = x µl Fugene6/ ____ µl Optimem _= ___ µl Fugene6 needed

4. Prepare DNA: For each well to be transfected, dilute the appropriate amount of CRE-Lucif OR Control-Lucif 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/µl (if none left, prepare another stock from the primary stock of 401.6 µg/ml).

Mastermix 1:
300 ng CRE-Lucif
3 ng Renilla plasmid

300 ng CRE-Lucif / 500 ng/ml = ____ µl Creb-Lucif plasmid
# of wells to receive CRE-Lucif: ____
____ * _____ µl Creb-Lucif plasmid _= _______ µl CRE-Lucif plasmid required for these wells

3 ng Renilla plasmid / 1 ng/ul = ____ µl Renilla plasmid
# of wells to receive Renilla (same as above): _____
____ * _____ µl Renilla plasmid _= _______ µl Renilla plasmid required for these wells

Mastermix 2:
300 ng Control-Lucif
3 ng Renilla plasmid

300 ng Control-Lucif / 500 ng/ml = ____ µl Creb-Lucif plasmid
# of wells to receive Control-Lucif: ____
____ * _____ µl Control-Lucif plasmid _= _______ µl Control-Lucif plasmid required for these wells

3 ng Renilla plasmid / 1 ng/µl = ____ µl Renilla plasmid
# of wells to receive Renilla (same as above): _____
____ * _____ µl Renilla plasmid _= _______ µl Renilla plasmid required for these wells
5. Add 100 µl 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.