/wp-content/uploads/2015/09/kuperwasser-banner-500px-wide-no-bg-semi-bold.png 0 0 admin /wp-content/uploads/2015/09/kuperwasser-banner-500px-wide-no-bg-semi-bold.png admin2015-09-02 19:44:082015-09-02 19:47:22Reading Luciferase and Renilla signal using Promega Dual Luciferase Reporter Assay
Dual Luciferase Reporter Assay System (Promega cat # E1910)
- Thaw frozen buffers in 37 C waterbath.
- Turn on luminometer to warm up before taking readings.
- Dilute Passive Lysis Buffer (PLB) 1:5 in distilled water and mix well.
- 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.
- 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
- Predispense 100 µl of LARII into appropriate number of eppendorfs to complete desired number of luciferase and Renilla readings.
- Check that luminometer is programmed to perform a 3 second premeasurement delay, followed by a 10 second measurement period for each assay.
- Aspirate media from cells and wash with PBS.
- Aspirate PBS and add appropriate amount of diluted PLB (see chart below) to plates (lysis).
- Lyse cells using a cell scraper.
- 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.
- If luminometer does not print, record the luciferase activity measurement by hand.
- Remove eppendorf from machine and add 100 µl Stop & Glo Reagent.
- Vortex sample for a few seconds.
- Place in luminometer again and take Renilla reading. Record by hand if luminometer does not print.
- Discard this tube, and repeat again with next sample.
- Calculate Luciferase/Renilla signal intensity, and normalize signal to that from cells transfected with a control-luciferase reporter plasmid and Renilla.
|6-well culture plate||500µl|
|12-well culture plate||250µl|
|24-well culture plate||100µl|
|48-well culture plate||65µl|
|96-well culture plate||20µl|