Arturo Casini:promchar

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Promoter strength characterisation routine, development version

• Arturo Casini 08:33, 18 November 2010 (EST):

Day before:

Set up cultures in 14 ml Falcon clip-top round bottom tubes, using 2-5 ml of medium with the appropriate antibiotic

For each sample, prepare 1 LB and 1 M9 culture (add medium producer/formula)

Incubate overnight, shaking at 37 degs

Day of the experiment:

Start early in the morning (around 9 a.m.)

Take the cultures out of the incubator

Load 100 ul of every culture on a 96 well plate (any) and measure the OD

Start new cultures in 14 ml Falcon clip-top round bottom tubes, using 2-5 ml of medium with the appropriate antibiotic so that they have a starting OD of 0.03 (possibly use 5 ml tubes with 2 ml culture instead?)

Incubate these cultures in a 37 degs shaking incubator for 1 hour 30 mins (possibly 1 hour?)

Check the OD at the end of the incubation using a 96 well plate again, and if they are different, adjust the volumes in the next step

Transfer 110 ul of the cultures on a Greiner flat bottom 96 well plate (black sides), filling three wells with each culture and using the wells on the side of the plate for media only

The plate will be incubated with a lid in a non-shaking 37 degs incubator for 5 hours

Using the plate reader (add the model) take GFP/RFP fluorescence measurements and OD measurements (check if we can use path length correction at 100 ul or how to adjust the OD calculation if we don’t) every hour for 5 hours, including timepoint 00:00. The measurement programs will shake the plate before taking the measurements.

Remember to adjust the gain before the first measurement and to write down the values.

Every 2.5 hours, after the plate reader measurements, take 5 ul of culture from each will with a multichannel pipette and transfer to a 96 well plate (any) that has been preloaded with uncontaminated distilled water (check the volumes, at least a 1/100 dilution is required, possibly more)

Load the plate on the cytometer (add model and specifications) and take GFP and RFP measurements (add settings)

Remember to adjust the gain before the first measurement and to write down the values.

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