Oct 16, 2019
OD and GFP Plate Reader Assay (72 h Measurement)
- 1Wageningen University
- iGEM Wageningen 2019
Protocol Citation: Alba Balletbó 2019. OD and GFP Plate Reader Assay (72 h Measurement). protocols.io https://dx.doi.org/10.17504/protocols.io.8aihsce
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: October 16, 2019
Last Modified: October 16, 2019
Protocol Integer ID: 28714
Abstract
Fluorescence measurements of Optical Density (OD) and Green fluorescent protein (GFP) in Escherichia coli.
Materials
MATERIALS
Microplate Reader Synergy Mx
Nunc™ MicroWell™ 96-Well Microplates, F 96 well plate, cell culture, clear, with lid, SterileThermo FisherCatalog #167008
Media Preparation
Media Preparation
Prepare the following media and autoclave them according to standard procedures.
M9TG Media (Without carbon source):
| Reagent | Ammount to add for 50 mL | |
| M9 Salts | 1X | |
| Tryptone | 0.5 g |
M9TG Media (0.5% Glycerol):
| Reagent | Ammount to add for 50 mL | |
| M9 Salts | 1X | |
| Tryptone | 0.5 g | |
| Glycerol | 0.25 g |
M9TG Media (2% Glycerol):
| Reagent | Ammount to add for 50 mL | |
| M9 Salts | 1X | |
| Tryptone | 0.5 g | |
| Glycerol | 1 g |
M9TG Media (0.5% Succinate and 1mM Leucine):
| Reagent | Ammount to add for 50 mL | |
| M9 Salts | 1X | |
| Tryptone | 0.5 g | |
| Succinate | 0.25 g | |
| Leucine | 6.56 mg |
Bacteria and Plate Reader Preparation
Bacteria and Plate Reader Preparation
Grow desired bacteria overnight in a 50 ml falcon tub, containing 10 mL M9TG media and grown cultures @
37 °C , 250 RPM overnight.
Note
Luria-Bertani media is also possible to use, but cells must be washed with PBS buffer prior to plate reader experiment because of the high amount of fluorescence from the yeast extract:
After overnight incubation, spin cells down at 4700 x G, for 5 min, discard the supernatant and resuspend in 10 ml PBS.
Repeat prior steps and resuspension again in PBS.
Load 190 µL of the corresponding buffer in the wells of the 96-well plate and add 10 µL of overnight culture.
Note
The amount of buffer and overnight culture will depend on the OD after overnight bacterial cultivation. The amounts present in this protocol where determined by an OD ~0.3.
Note
When cell have been grown overnight in LB media, washing steps are require before continuing! (See step 3).
Plate Reader Protocol
Plate Reader Protocol
Set the following variables for the BioTek Synergy MX Microplate Reader:
- Temperature: Setpoint 37°C
Preheat before moving to next step. Start Kinetic [Run 72:00:00, Interval 0:04:20]
- Shake: Medium for 0:05 (MM:SS)
- Read OD600:
Absorbance Endpoint of full plate
Wavelengths: 600
Read Speed: Normal
- Read GFP:
Fluorescence Endpoint of full plate
- Filter Set:
Excitation: 488/9,0
Emission: 510/9,0
Optics: Bottom
Gain: 100
Read Speed: Normal
Start plate reader protocol.
Data Analysis
Data Analysis
Retrieve the data from the computer, correct for the measurements for the OD600 and subtract the autofluorescence from the control sample of all the other samples.