• Reagents:

Dual Luciferase Reporter Assay System (Promega cat # E1910)

Eppendorfs

Luminometer

Sample plate(s)

PBS

Distilled water

Cell scrapers


• Preparation:

1. Thaw frozen buffers in 37 C waterbath.
2. Turn on luminometer to warm up before taking readings.
3. Dilute Passive Lysis Buffer (PLB) 1:5 in distilled water and mix well.
4. Prepare Luciferase Assay Reagent II (LARII) by adding 10 ml Luciferase Assay Buffer II to lyophilized substrate. Aliquot LARII in eppenndorfs and store unused aliquots at -80 (stable for up to 1 yr) protected from light.
5. Prepare an adequate volume of Stop & Glo Reagent to perform desired number of assays. Dilute Stop & Glo Substrate 1:50 with Stop & Glo Buffer in a 15 ml falcon tube.
   100 µl Stop & Glo Reagent/assay * _ assays (aka readings) = __ µl Stop & Glo needed + __ µl pipet error = ____ µl total volume of Stop & Glo Reagent needed
6. Predispense 100 µl of LARII into appropriate number of eppendorfs to complete desired number of luciferase and Renilla readings.
7. Check that luminometer is programmed to perform a 3 second premeasurement delay, followed by a 10 second measurement period for each assay.

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   NOTE: these are default settings on the Kuperwasser luminometer).

• Protocol:

1. Aspirate media from cells and wash with PBS.
2. Aspirate PBS and add appropriate amount of diluted PLB (see chart below) to plates (lysis).
3. Lyse cells using a cell scraper.
4. Transfer 20 µl of lysis sample to eppenndorf tube containing LARII, mix up and down with a pipet 2-3 times. Place tube in luminometer and take reading.
5. If luminometer does not print, record the luciferase activity measurement by hand.
6. Remove eppendorf from machine and add 100 µl Stop & Glo Reagent.
7. Vortex sample for a few seconds.
8. Place in luminometer again and take Renilla reading. Record by hand if luminometer does not print.
9. Discard this tube, and repeat again with next sample.
10. Calculate Luciferase/Renilla signal intensity, and normalize signal to that from cells transfected with a control-luciferase reporter plasmid and Renilla.