TITLE: Absorbance assay from an M2P flower plate
AUTHORS: Tijana Radivojevic, Matthew Incha, Apostolos Zournas, vblayroger, Stephen Tan, Hector Garcia Martin

[DESCRIPTION]
This protocol explains the steps for preparing samples and taking OD600 and OD340 measurements from a BioLector plate.



[BEFORE_START]
Make sure you booked the equipment in the calendar:
978-4-BIOMEK-NX-S8_ SPECTRAMAX (4148) EQ (1) (or any other "plate-reader" capable of measuring OD340 and OD600)
978-4-BIOMEK-NXP (4148) EQ (1)

Make sure you received proper training before operating on the Biomeks. 





[STEPS]
SECTION: Required equipment/labware
1. Destination plate:

 

Water plate:
 
Supernatant plate:
 
 

SECTION: Required equipment/labware
2. Liquid handler:
 

Pipette tips needed (available at Robotics lab):
tips s200 (green box)
tips p1000 (yellow box)

Spectrophotometer/plate-reader:
Molecular Devices SpectraMax M2
or any other spectrophotometer capable of measuring OD340 and OD600

SECTION: Required equipment/labware
3. Additional components:
H2O
m2p plate with cultures

Centrifuge options:
Avanti-15R-centrifuge
Allegra-25R
Eppendorf 5810R
(any swinging-bucket centrifuge capable of spinning a 96-deepwell plate)
NOT the Eppendorf 5430


SECTION: Biomek setup
4. 
 

We will use two Biomek methods for this assay.

The first method will prepare:
a Tecan plate for OD600 measurements, with 10x dilution of the samples  
two 96-deepwell plates as an intermediate step for preparing a Tecan plate for OD340 measurements - one with 1 mL of samples (supernatant plate) and the other with 1 mL of water for balance in the centrifuge 

The second method will prepare:
a Tecan plate for OD340 measurements (aliquoting cell supernatants from the 96-deepwell plate)
SECTION: Biomek setup
4.1. Open the Biomek Software application on the Biomek. At the top toolbar, select Project > Open Project to open the "Automation Dev" project.
 
 

SECTION: SpectraMax run
6. Start the Spectramax software "SoftMax"
Click "Open a data file" in the top left of the SoftMax window
Open the method for measuring OD600 named OD600.sda in the "My_Documents/Keasling_Lab/inch" subfolder on the Spectramax (NX-S8) computer

 
 
Open tray and place plate
Measure OD600 from the first prepared 96-well Tecan plate
Save the data (see step 7)

Click "Open a data file"
Open a method for measuring OD340 named OD340.sda in "My_Documents/Keasling_Lab/inch" subfolder on the Spectramax (NX-S8) computer
Measure OD340 from the second prepared 96-well Tecan plate
Save the data (see step 7)


SECTION: Saving data
7. Copy the values for OD600 from the SoftMax window into an empty Excel file and rename the spreadsheet to "600"
Copy the values for OD340 to another spreadsheet of the same file and rename to "340"
Save the Excel file as "OD.xlsx"

 

SECTION: Biomek setup
4.2. At the top toolbar of the Biomek Software, select File > Open to open the first method.
 
To open the second choose:
 

SECTION: Biomek run
5. 
 

Hit the play button (green arrow) to run the method.
 

SECTION: Biomek run
5.1. The first method takes around 5 min before the pause and 5 min after the pause
 
When the method pauses, remove the two 96-deepwell plates and continue the Biomek run
 
SECTION: Biomek run
5.2. Centrifuge run:

Place the two 96-deepwell plates sealed in the centrifuge and spin at max speed for 5 min 
SECTION: Biomek run
5.3. Carefully remove the supernatant plate from the centrifuge, remove the seal and place it back in the Biomek 
Start the second Biomek method to prepare a Tecan plate for OD340 measurements (takes around 5 min )