Goal: Determine chlorophyll-a in Astrangia samples from 2021 experiment

This protocol was adapted from the Putnam lab, Emma Strand, and Wall et al. 2018.

Materials & Equipment

• 100% acetone
• Quartz 96 well plate
• Microcentrifuge
• P1000 + tips
• 1.5 mL tubes
• Microplate reader
• Gen5 software

Protocol

1. Thaw sample homogenate
2. Centrifuge the aliquot of adult homogenate for 3 minutes at 13,000 rpm to separate host and symbiont cells.
3. Remove and discard supernatent. The pellet is the symbiont cells!
4. Add 1 mL of 100% acetone to the pellet. Vortex the tubes for 15 seconds to mix.
   • I also ‘beat’ the samples (i.e., shook them really fast on the Benchmark), as I wanted to dissolve the symbiont pellet as much as I could.
5. Put tubes in the 4°C fridge in the dark for 24 hours.
6. Make a plate map for samples.
7. The following day, take the tubes out of the fridge and vortex for 15 seconds.
8. Spin tubes for 3 minutes at 13,000 rpm.
9. Using the quartz 96 well plate, pipette 200 µL of 100% acetone into duplicate wells to be used as blanks.
10. Pipette 200 µL of sample to duplicate wells of 96-well quartz plate.
11. Measure the extracted cholophyll-a concentration on the plate reader at 630, 663, and 750 nm.

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