De la Cruz:Victor3 Multiplate reader

GENERAL PROTOCOL FOR EXPRESSION PROFILING (GFP)

1.- Initial Growth

We use 96 well plates (500 μL capacity each well). Do not fill completely the well, leave at least ½ of the well empty for better aireation. Seal the plate with a gas-permeable membrane. Grow the cells at 37ºC with orbital shaking in M9 medium supplemented with 0.4% glycerol, 0.2% casaminoacids and appropriate antibiotics. (16-22 hours)

2.- Measurement

Dilute cells 1/10000 in the same media and add 150 to a 96 well plate for measurement. The measurement protocol in the Victor3 is

-OD600 measurement -GFP measurement -Shaking

-OD600 measurement -GFP measurement -Shaking

-OD600 measurement -GFP measurement -Shaking

Water injection (use Mq water!) to counteract evaporation. This step must be adjusted if you other temperatures than 37ºC. For this temperature the evaporated volume is about 6 . DO NOT USE when measuring at 25ºC or 42ºC since evaporation will make your growing medium to dilute/evaporate. Calculate the rates of evaporation for these temperatures.

Each shaking interval takes about 6 minutes and the complete sequence takes 20 min. Standard measurement repeats the routine 40 times. This takes about 15 hours.

VICTOR3 SETUP:

OD600 label Absorbance=Visible OD600 filter in SlotA7; Reading time= 0.5 sec.

GFP label CW Energy = 10320 (Stabilized) CW-Filter = F485 SlotA5 Emission Filter= F535 SlotA5 Counting time= 0.1 sec.

Shaking Duration 180 sec Speed= Normal Type= Orbital