Team:WHU-China/Calibration

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Calibration

• 1 OD600 Reference point
• 2 FITC fluorescence standard curve

OD600 Reference point

• Add 100 μl LUDOX into wells A1, B1, C1, D1 (or 1 mL LUDOX into cuvette)

• Add 100 μl of H2O into wells A2, B2, C2, D2 (or 1 mL H2O into cuvette)

• Measure Abs600 of all samples in all standard measurement modes in instrument

• Record the data

• Import data into Excel (OD600 reference point tab) Sheet_1 provided

FITC fluorescence standard curve

#Prepare the FITC stock solution:

• Spin down FITC stock tube to make sure pellet is at the bottom of tube

• Prepare 2x fluorescein stock solution (100µM) by resuspending fluorescein in 1mL of 1xPBS.

• Dilute the 2x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution and resulting concentration of fluorescein stock solution 50µM (500µL of 2x fluorescein in 500 µL 1x PBS will make 1 mL of 50 µM (1x) fluorescein solution.)

#Prepare the serial dilutions for fluorescein:

• Add 100 µl of PBS​ into wells A2, B2, C2, D2....A12, B12, C12, D12

• Add 200 µl​ ​of FITC 5x stock​ solution into A1, B1, C1, D1

• Transfer 100 µl of FITC stock solution from A1 into A2.

• Mix A2 by pipetting up and down 3x and transfer 100 µl into A3…

• Mix A3 by pipetting up and down 3x and transfer 100 µl into A4...

• Mix A4 by pipetting up and down 3x and transfer 100 µl into A5...

• Mix A5 by pipetting up and down 3x and transfer 100 µl into A6...

• Mix A6 by pipetting up and down 3x and transfer 100 µl into A7...

• Mix A7 by pipetting up and down 3x and transfer 100 µl into A8...

• Mix A8 by pipetting up and down 3x and transfer 100 µl into A9...

• Mix A9 by pipetting up and down 3x and transfer 100 µl into A10...

• Mix A10 by pipetting up and down 3x and transfer 100 µl into A11...

• Mix A11 by pipetting up and down 3x and transfer 100 µl into ​liquid waste

• Repeat dilution series for rows B, C, D

• Measure fluorescence of all samples in all standard measurement modes in instrument

• Record the data

• Import data into Excel (FITC standard curve tab) Sheet_1 provided