#5


LUCIFERASE ASSAY AND BETA GAL ASSAY AFTER TRANSFECTION OF EUKARYOTIC
CELLS USING A TUBE LUMINOMETER


1.      Collect cells
--From a 24 well, set up eppies, and collect ~1ml media into eppie.
--Then, wash briefly with ~250ul 1xPBS  place in the same eppie
--Then, add ~250ul trypsin (~4-5 drops with 5ml pipette)  15min 37C incubator
--At the end of Trypsin incubation, you can use the solution
(Media/PBS) present in the eppie to "wash" the well, and place back in
eppie. So, at the end, you have collected all cells of a certain well
into 1 eppie, ~1.5ml at the end.
--Spin eppies at 1500 rpm 5min, aspirate supernatant carefully with
10ul white pipette attachment to vaccum.

2.      Add 50ul per eppie of 1x passive lysis buffer (also called reporter
lysis buffer). Stock is at RT and is 5x.

3.      Put eppies in a foam shaker and shake at speed 3 for 15 minutes

4.      Freeze the eppies at –40C or –20C (at least 30min)

5.      Thaw briefly, vortex once more at high speed and spin in microfuge
at 13,000rpm or max speed for 30seconds.

6.      Place tubes on ice = LYSATES

7.      LUC ASSAY:

a.      Thaw the Luc substrate in Amber bottle.  Place on ice.
b.      Use 50ul of luc substrate per reading.
c.      Place new eppies on a rack, add 50ul Luc substrate to each
d.      Go to luminometer area, add to Substrate eppie, 20ul of each lysate.
e.      Vortex briefly high speed and read in tube luminometer
Settings: Luc-0-Inj program (10sec readings)

8.      If your assay was a transfection, do also Beta-gal kit assay
(transfection efficiency control)

BETA GAL ASSAY:
a.      Prepare for each sample you need, a mix with: Bgal MIX=49ul Bgal
buffer and 1ul Bgal substrate
b.      Prepare a rack with new eppies, add 45ul of the Bgal MIX to each.
c.      Then, add 5ul of the LYSATE. Vortex on high, place in the dark
(closet or drawer) for 1 hour
d.      After 1 hour incubation at RT, read using luminometer setting (1 sec reading)