Adult coral Homogenate prep and symbiont separation

Background

This protocol is used to separate coral tissue slurry that is homogenized into host (coral) and symbiont (Symbiodinaceae) fractions. The samples used in this protocol were from the Astrangia poculata nutrition experiment run by K. Wong in April 2019. Corals were snap frozen, airbrushed with filtered seawater, homogenized with a glass homogenizer, aliquoted appropriately (500 μL in each tube), and frozen at -80°C. This homogenate separation will be used for citrate synthase analyses and DNA extractions

Equipment and reagents needed

  • Ice bucket
  • Lysis buffer
  • Wash buffer
  • Vortex
  • Centrifuge that can maintain 4°C

Reagent preparation

Lysis buffer

  • Can be found in this protocol
  • To make 250 mL of lysis buffer, add:
    • 6.25 mL 1 M Tris (pH 8)
    • 0.5 mL 0.5 M EDTA
    • 25 mL 100% glycerol [v/v]
    • 218.25 mL DI water
  • Store at 4°C

Wash buffer

  • 250 mL of lysis buffer
  • 25 μL of 0.01% [v/v] Triton X
  • Store at 4°C

Protocol

All steps must be performed on ice or at 4°C

  1. Thaw samples on ice and add 0.6mL of ice-cold lysis buffer
  2. Vortex samples and check cell integrity on a hemocytometer
  3. Centrifuge at 6000g for 2 mins at 4°C
  4. Remove 800 μL of supernatant and transfer to a new labelled tube (host fraction)
  5. Place symbiont pellet on ice

— HOST FRACTION —

  1. Centrifuge host fraction at 16000g for 20 minutes at 4°C to remove any particulate debris
  2. Transfer supernatant to a new tube and vortex
  3. Aliquot appropriately and store at -80°C for further analyses
    • e.g. 600 μL for DNA extractions, 100 μL for citrate synthase, and 50 μL for protein.

— SYMBIONT FRACTION —

  1. Resuspend pellet in 200 μL of lysis buffer
  2. Vortex and centrifuge at 700g for 5 minutes at 4°C
  3. Remove and discard supernatant
  4. Resuspend pellet in 1 mL of ice-cold wash buffer
  5. Repeat steps 10-12 for a total of 5 times
  6. Resuspend pellet in 300 μL of lysis buffer and add and 200 μL of 0.5mm diameter glass beads
  7. Vortex for 3 minutes
  8. Remove supernatant and aliquot accordingly
    • e.g. 200 μL for DNA extractions, 50 μL for citrate synthase, and 50 μL for protein
Written on April 24, 2019