Mix 1 to 30 ug DNA including Luciferase reporter and LacZ in 0.4 ml of RPMI/20%
FCS.
Spin down 5 X 10(6) B cells at 1 K for 5 min.
Resuspend cells in 0.4 ml DNA/RPMI/20% FCS.
Transfer cells into 0.4 cm Cuvette.
Incubate on ice for 10 min.
Electroporate at 250 Volts/960 uF, or 300 volts/500 uF.
(use Mammalian set-up (EXT), press both buttons until beep sounds.)
Set on ice for 5 min.
Transfer all cells into T25 flask.
Add 5 ml of RPMI complete medium.
Incubate for 24 to 48 hours at 37 C before assays.
If required, cell can be treated with stimuli at about 8 hours before assays.
Spin down cells at 1 K for 5 min.
Wash with 1 X PBS.
Add 300 ul of 1X Reporter lysis buffer (Promega).
Use P1000 to pipet all cells in each well into a new 1.5 ml microcentrifuge
tube.
Votex the cells.
Wait for 5 minutes at RT.
Put cells in - 80 C freezer and wait till all the cell lysates are frozen.
Thaw the cell lysates in 37 C waterbath till all ice are melted (DO NOT OVER
THAW).
Spin the cell lysates in the microcentrifuge for 2 minutes at 14K rpm.
Take 20 ul for luciferase assay.
Reagent
Z Buffer: Per liter
16.1 g Na2HPO4.7H2O
5.5 g NaH2PO4.H2O
0.75 g Kcl
0.246 g MgSO4.7H2O
Z buffer with b-2ME : mix 27 ul of b-2ME with 10 ml of Z buffer.
ONPG (O-Nitrophenyl-b-D-galactopyranoside): 4 mg/ml in H2O
1 M Na2CO3
Spin down the cell lysate.
Put 50 ul of cell lysate supernatant into a microcentrifuge tube.
Add 500 ul of Z buffer with b-2 ME.
Add 100 ul of ONPG (4 mg/ml) and Vortex.
Incubate the tubes at 30 C till you see yellow color.
Stop the reaction by add 250 ul of 1 M Na2CO3
Read at OD=420 (visible light).