De la Cruz:Victor3 Multiplate reader

RESPONSIBLE FOR VICTOR 3

Irene del Campo [[1]]

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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

VICTOR3 Clinical History

15-Feb-2008 Several errors appeared days ago. The errors seemed to be related to shaking, but the machine was able to continue measuring

26-Feb-2008 The errors continue, now the machine stops measuring calling for Error 4004. The technician was called.

07-Mar-2008 The technician came and he says that the motor is failing. He has loosen the belts and the machine now measures again, but he says that the motor must be changed.

• We have noticed that this is in fact the same error that appeared to Endy group in their Victor3. Seems that extensive shaking damages the machine. However we are using "normal" instead of "high" shaking and orbital instead of "double orbital".

The same day the technician came, the bulb burnt. Replaced.