Chlorophyll Protocol
Quantifying Chl-a and Chl-c2 Concentration in Symbiodiniaceae from Madracis decactis and Diploria labyrinthiformis tissue
This protocol was adapted from the E5 Protocol edited by the putnam lab, the Putnam lab, Emma Strand, and Wall et al. 2018.
Contents
- Materials
100% acetone
- flammable safe fridge 4°
- quartz 96 well plate
- Microcentrifuge (Mo’orea Gump Molecular Lab)
- 1ml pipette and tips
- 1.5 ml microfuge tubes
- Synergy HTX Multi-Mode Microplate Reader
- Gen5 Software
- Protocol
Sample Preparation
- Thaw homogenate aliquot.
- vortex sample for 15 seconds.
- If not done already, centrifuge the 500 μL aliquot of adult airbrush homogenate at 13,000 rpm for 3 minutes to separate the host and Symbiodiniaceae cells.
- Remove and discard the supernatant. The pellet is the symbiont cells!
- Add 1 mL of 100% acetone to the pellet in the 1.5 mL microcentrifuge tube and vortex the tubes to mix. (The orginal protocol say to vortex for 15seconds however it take more than 15 seconds to attempt to get the pellet to dissovle as much as possible. note: the entire pellet will not disolve during votexing but should once left over night.)
- Place the tubes in a fridge in the dark at 4°C for 24 hours.
- Vortex the tubes for 15 sec.
- Spin the tubes down at 13,000 rpm for 3 minutes in the microcentrifuge to pellet any debris.
- Pipette 200µl of sample to duplicate wells of 96-well quartz plate.
- Pipette 200µl of acetone blank to duplicate wells.
- Cover the plate with silicone pad every 5th sample or so to reduce evaporate as samples are added.
- Remove silicon pad.
- Follow steps below to measure the extract Absorbance on the Synergy HTX Multi-Mode Microplate Reader at 630, 663, and 750 nm in a 96-well quartz plate.
- Standardize for path length in 200µl of sample in 96-well quartz plate.
Measure the Absorbance 1.Open the Gen5 software on your computer. 2.Once Gen5 opens on the computer you can select the pre-made protocol from the drop down menu. 4.The plate loader should automatically open.Once the software is open on the computer. 5.Place the 96 quart plate in the plate reader. 5.To measure Absorbance, select the pre-made ‘Chlorophyll Protocol’ option and press the green start protocol button and allow to run through the 630,663 and 750nm. 6.Export the raw GEN5 software data file and a CSV to the Desktop in a designated folder.
Calculating Chlorophyll Concentration Chlorophyll a and c2 concentrations are calculated from the equations in Jeffrey and Humphrey 1975 after substracting A750nm from all measurements.
Need to correct for differences in path length of the volume in the 96 well plate compared to the 1cm path length of a cuvette. Warren 2007
R code for this analysis can be found at K. Wong’s Github.