May 06, 2019
Calibration Protocol - Fluorescence Standard Curve with Fluorescein
- 1iGEM Measurement Committee
- iGEM MeasurementTech. support email: pauljrutten@gmail.com
External link: https://2019.igem.org/Measurement
Protocol Citation: Richard Tennant, Paul Rutten 2019. Calibration Protocol - Fluorescence Standard Curve with Fluorescein. protocols.io https://dx.doi.org/10.17504/protocols.io.zgkf3uw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 24, 2019
Last Modified: August 07, 2019
Protocol Integer ID: 21740
Abstract
Plate readers report fluorescence values in arbitrary units that vary widely from instrument to instrument. Therefore absolute fluorescence values cannot be directly compared from one instrument to another. In order to compare fluorescence output of test devices between teams, it is necessary for each team to create a standard fluorescence curve. Although distribution of a known concentration of GFP protein would be an ideal way to standardize the amount of GFP fluorescence in our E. coli cells, the stability of the protein and the high cost of its purification are problematic. We therefore use the small molecule fluorescein, which has similar excitation and emission properties to GFP, but is cost-effective and easy to prepare. (The version of GFP used in the devices, GFP mut3b, has an excitation maximum at 501 nm and an emission maximum at 511 nm; fluorescein has an excitation maximum at 494 nm and an emission maximum at 525nm).
You will prepare a dilution series of fluorescein in four replicates and measure the fluorescence in a 96 well plate in your plate reader. By measuring these in your plate reader, you will generate a standard curve of fluorescence for fluorescein concentration. You will be able to use this to convert your cell based readings to an equivalent fluorescein concentration. Before beginning this protocol, ensure that you are familiar with the GFP settings and measurement modes of your instrument. You will need to know what filters your instrument has for measuring GFP, including information about the bandpass width (530 nm / 30 nm bandpass, 25-30 nm width is recommended), excitation (485 nm is recommended) and emission (520-530 nm is recommended) of this filter.
Note: The iGEM Particle Standard Curve with Microspheres calibration method is a pre-requisite for carrying out this protocol. You will need data from that calibration to analyse the results of this protocol.
Attachments
Materials
MATERIALS
96 well plate
PBS
Fluorescein
STEP MATERIALS
PBS
Fluorescein
Fluorescein is provided in the iGEM Measurement Kit. The 96-well plate should preferably be black with a clear flat bottom.
Protocol materials
96 well plate
PBS
Fluorescein
PBS
Fluorescein
Fluorescein
PBS
Before start
Read through this entire protocol carefully before you start your experiment and
prepare any materials you may need.
Note: The iGEM Particle Standard Curve with Microspheres calibration method is a pre-requisite for carrying out this protocol. You will need data from that calibration to analyse the results of this protocol.
Prepare the fluorescein stock solution
Prepare the fluorescein stock solution
Spin down fluorescein kit tube to make sure pellet is at the bottom of tube
Fluorescein
Prepare 10x fluorescein stock solution (100 μM) by resuspending fluorescein in 1mL of 1X PBS
Note
It is important that the fluorescein is properly dissolved. To check this, after the resuspension you should pipette up and down and examine the solution in the pipette tip – if any particulates are visible in the pipette tip continue to mix the solution until they disappear.
PBS
Dilute the 10X fluorescein stock solution with 1X PBS to make a 1X fluorescein solution with concentration 10 μM: 100 μL of 10X fluorescein stock into 900 μL 1X PBS
Prepare the serial dilutions of fluorescein
Prepare the serial dilutions of fluorescein
Accurate pipetting is essential. Serial dilutions will be performed across columns 1-11. Column 12 must contain PBS buffer only. Initially you will setup the plate with the fluorescein stock in column 1 and an equal volume of 1X PBS in columns 2 to 12.
You will perform a serial dilution by consecutively transferring 100 μl from column to column with good mixing.
Add 100 μl of 1X PBS into wells A2, B2, C2, D2....A12, B12, C12, D12
Add 200 μl of fluorescein 1X stock solution into A1, B1, C1, D1
Transfer 100 μl of fluorescein 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
Note
Take care not to continue serial dilution into column 12
Repeat dilution series for rows B, C, D
Measure fluorescence
Measure fluorescence
Measure fluorescence of all samples in instrument
Record the data in your notebook
Import data into this Excel sheet provided (fluorescein standard curve tab): 
iGEM Data Analysis Template - Fluorescence Standard Curve - v1.xlsx
iGEM Data Analysis Template - Fluorescence Standard Curve - v1.xlsx Congratulations!
Congratulations!
You have now completed this calibration protocol