Sep 27, 2020
Version 2
Calibration Protocol - Plate Reader Abs600 (OD) Calibration with Microsphere Particles V.2
Version 1 is forked from Calibration Protocol - Particle Standard Curve with Microspheres
- Jacob Beal1,
- Traci Haddock-Angelli2,
- Markus Gershater3,
- Vishal Sanchania3,
- Russell Buckley-Taylor1,
- Geoff Baldwin4,
- Natalie Farny1,
- Richard Tennant1,
- Paul Rutten1
- 1iGEM Measurement Committee;
- 2iGEM;
- 3Synthace;
- 4Imperial College London
- iGEM MeasurementTech. support email: pauljrutten@gmail.com
External link: https://2019.igem.org/Measurement
Protocol Citation: Jacob Beal, Traci Haddock-Angelli, Markus Gershater, Vishal Sanchania, Russell Buckley-Taylor, Geoff Baldwin, Natalie Farny, Richard Tennant, Paul Rutten 2020. Calibration Protocol - Plate Reader Abs600 (OD) Calibration with Microsphere Particles. protocols.io https://dx.doi.org/10.17504/protocols.io.549g8z6
Manuscript citation:
Fedorec AJ, Robinson CM, Wen KY, Barnes CP, FlopR: An Open Source Software Package for Calibration
and Normalization of Plate Reader and Flow Cytometry Data. ACS Synthetic Biology 9(9). doi: 10.1021/acssynbio.0c00296
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: August 05, 2019
Last Modified: September 27, 2020
Protocol Integer ID: 26497
Abstract
You will prepare a dilution series of monodisperse silica microspheres and measure the Abs600 in your plate reader.
The size and optical characteristics of these microspheres are similar to cells, and there is a known amount of particles per volume. This measurement will allow you to construct a standard curve of particle concentration which can be used to convert 600 nm absorbance measurements into an estimated equivalent number of cells.
Attachments
Guidelines
For a full set of calibrations, you should run two protocols: this Abs600 (OD) calibration with microspheres, and the fluorescence calibration curve with fluorescein.
Before beginning these protocols, please ensure that you are familiar with the measurement modes and settings of your instrument. For all of these calibration measurements, you must use the same plates and volumes that you will use in your cell-based assays. You must also use the same settings (e.g., filters or excitation and emission wavelengths) that you will use in your cell-based assays. If you do not use the same plates, volumes, and settings, the calibration will not be valid.
Make sure to record all information about your instrument to document your experiment. If your instrument has variable temperature settings, the instrument temperature should be set to room temperature (approximately 20-25 C) for all measurements.
Materials
MATERIALS
96 well plate
double distilled water (ddH2O)
300µl Silica beads
STEP MATERIALS
300µl Silica beads
ddH20
300 μL Silica beads are provided in the iGEM Measurement Kit. The 96-well plate should preferably be black with a clear flat bottom.
Protocol materials
96 well plate
double distilled water (ddH2O)
300µl Silica beads
300µl Silica beads
ddH20
300µl Silica beads
ddH20
Before start
Read through this entire protocol carefully before you start your experiment and prepare any materials you may need.
Prepare the Microsphere Stock Solution
Prepare the Microsphere Stock Solution
Obtain the tube labeled “Silica Beads” from the Measurement Kit and vortex vigorously for 30 seconds.
300µl Silica beads
Note
Microspheres should NOT be stored at 0°C or below, as freezing affects the properties of the microspheres. If you believe your microspheres may have been frozen, please contact the iGEM Measurement Committee for a replacement (measurement@igem.org).
Immediately pipet 100 μL microspheres into a 1.5 mL eppendorf tube
Add 900 μL of ddH2O to the microspheres
ddH20
Vortex well. This is your Microsphere Stock Solution
Prepare the serial dilution of microspheres
Prepare the serial dilution of microspheres
Accurate pipetting is essential. Serial dilutions will be performed across columns 1-11. Column 12 must contain ddH2O only.
Initially you will setup the plate with the microsphere stock solution in column 1 and an equal volume of 1x ddH2O 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 ddH2O into wells A2, B2, C2, D2....A12, B12, C12, D12
Vortex the tube containing the stock solution of microspheres vigorously for 10 seconds
Immediately add 200 μl of microspheres stock solution into A1
Transfer 100 μl of microsphere 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
IMPORTANT!
Re-Mix (pipette up and down) each row of your plate immediately before putting in the plate reader! (This is important because the beads begin to settle to the bottom of the wells within about 10 minutes, which will affect the measurements.)
Note
Take care to mix gently and avoid creating bubbles on the surface of the liquid
Measure OD
Measure OD
Measure OD600 of all samples in instrument. Disable any path length correction setting on your instrument, if it has one.
If you will be using your data in conjuction with measurements from the Fluorescence standard curve protocol, make sure you use the same instrument settings for both protocols.
Record the data in your notebook. Please note your standard curve should still work well even if a few of your measurements are saturing the instrument
Import data into this Excel sheet:
iGEM Data Analysis Template - Particle Standard Curve - v1.xlsx Congratulations!
Congratulations!
You have now completed this calibration protocol