Yeast growth and fluorescence 96-well plate reader experiment, set up from a 96 deep well plate

Plan the plate set up for the experiment, both the 1) pre-culture plate and 2) the final 96-well black microtiter plate.

Tips for plate design:

• For the pre-culture plate, fill the plate, skipping every other row to allow a checkered pattern of loading
• Plan the final plate set up in blocks, where each block (labelled in black and red thick lines the final plate) is a technical replicate. This allows blocks to be set up in different ways, e.g. upside down.

  

Aliquot 100µL of filter sterilised yeast media (with selection as needed) to each well to be used for the experiment according to the pre-culture plate plan.

This can be done by two methods:

1. Using a multi-channel pipette, with the media in a reagent reservoir.

2. Using a repeat pipette.

Seal with a gas permeable seal. If the plate was designed with the outer wells empty, add 500µL of ddH2O.

Grow overnight in a incubator set at 30°C and shaking at 250 rpm.

Note: 250rpm is a minimum! Otherwise yeast cells will pellet instead of growing in suspension.

After 20 hours, carefully add 900µL of yeast media to each well containing yeast culture (this is a 1:10 dilution of the culture)

This can be done by two methods:

1. Using a P1000 multi-channel pipette, with the media on a reagent reservoir.

2. Using a repeat pipette.

Using a P100 multi-channel pipette, resuspend the culture several times to mix then transfer 200µL of the culture to a clear 96 well microtiter plate.

Measure the OD600 using the plate reader.

Blank correct all the OD measurements by subtracting all the measured OD by the OD of the media.

On a Tecan M200 plate reader, this can be done in (a copy of) the excel spreadsheet that contains the output measurements.

Apply the following formula to each blank corrected OD measurement. The result of this will be the amount to add to the diluted pre-culture to normalise their OD

=800 * (( x - target OD ) / target OD)

• 800 is for the remaining volume of the culture on the pre-culture deep well plate
• x is for the blank corrected OD value

Add the volumes of media as calculated from the previous step to their corresponding wells to normalise their ODs.

Using a multi-channel pipette, add 200µL of media to the (blank) media wells of the black microtiter plate.

Using a multichannel pipette, gently resuspend the diluted culture by pipetting up and down several times, before loading them on the next plate.

Load 200µL of diluted culture to each well of the black microtiter plate, following the plate design. Load one biological replicate at a time and repeat 3x for technical replicates before moving to the next biological replicate.

Replace the lid and transfer the plate to the Tecan M200 plate reader.

On the Tecan plate reader program (Tecan icontrol), set up the following protocol:

• Select the plate you are using - (Corning flat bottom 96 black microtiter plate).

• Select the wells you would like to measure.

• Set the temperature at 29.9-30.5 ℃.

• From the actions/functions on the left side of the screen, add a 'kinetic cycle' to the program. - Set this to 80 cycles, linear shaking, 6 mm amplitude at 200-220 rpm, for a duration of 550 seconds.

• Add an absorbance measurement to the kinetic cycle. - Set this to 595 nm with a maximum bandwidth of 9 nm with 15 reads.

• If needed, add a fluorescence measurement to the kinetic cycle - For mCherry fluorescence - set this to an excitation wavelength at 585 nm and

            an emission wavelength of 620 nm (excitation bandwidth of 9 nm and 
  
            emission bandwidth of 20 nm) with the gain set at 100. 
  
        - For mTurquoise2 fluorescence - set this to an excitation wavelength at 434 nm 
  
            and an emission wavelength of 474 nm (excitation bandwidth of 9 nm and 
  
            emission bandwidth of 20 nm) with the gain set at 60. 
  

The steps included in the kinetic cycle are indented.

Start the program. The plate reader should begin to heat up to ~30℃. After the temperature is reached, the plate reader should begin the shaking step. Wait until the first absorbance and fluorescence measurement is recorded to make sure the protocol is running as planned, before leaving the experiment to run.

When the experiment has finished running, save the raw data file from the Tecan icontrol software.

Give this data file a sensible name including the date of the experiment in a standard format (e.g. 2021-08-10). Backup the raw data file in the lab's shared storage space and make a copy for any data analysis, do not overwrite your raw data! Link to both the raw data and the analysis in your lab notebook entry.