At indicated dates, individual corals were visually assessed for bleaching. Bleaching was noted for those corals paling or entirely lacking in pigmentation.\u00a0 A reference color card (Seibeck et al., 2006) was used as a pigmentation reference. The normal level of pigmentation was C3 or darker (not bleached). Colors lighter than C3 were considered bleached. Using bone cutters, a small sub-apical piece was broken off of each coral. On the boat, samples were transferred to CHAOS buffer (listed below) and extracted for DNA:
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Chaos Buffer:
\n
4.5M Guanidinium thiocyanate
\n2% N-lauroylsarcosine (sarcosyl)
\n50mM EDTA
\n25mM Tris-HCl, pH 7.5
\n0.2% antifoam A (Sigma)
\n0.1M b-mercaptoethanol (BME)
\n
Binding buffer (BB):
\n
6 M GuSCN 1 , (Guanidine Thiocyanate)
\n20 mM EDTA pH 8.0
\n10 mM Tris-HCl pH 7.5
\n4% Triton X-100
\n
Protein wash buffer (PWB):
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70 mL of ethanol (96%) was thoroughly mixed with 26 mL of BB (stable at 20 deg\u00a0C for1 week).
\n
Wash buffer (WB):
\n
ethanol (60%),
\n50 mM NaCl
\n10 mM Tris-HCl pH 7.4
\n0.5 mM EDTA pH 8.0 (stored at -20 deg\u00a0C)
\n
-Allow coral fragments to sit covered in CHAOS buffer in 1.5 - 2.0 mL tube for days- weeks.
\n-Pipet the Supernatant mixture into a silica spin column placed in a 2 ml collection tube. Centrifuge at 6000 x g (8000 rpm) for 1 min. Discard flow-through. Repeat as needed to load entire sample.
\n-Add 700 ul PWB to column, and centrifuge for 1 min at 6000 x g (8000 rpm). Discard flow-through.
\n-Repeat 2x (3 washes total).
\n-Add 700 ul WB to column and centrifuge for 1 min at 20,000 x g (14,000 rpm). Discard flow-through.
\n-Repeat wash and centrifuge for 3 min at 20,000 x g (14,000 rpm) to dry the DNeasy membrane.
\n-Discard flow-through and collection tube.
\n-Place the spin column in a clean 1.5 ml or 2 ml microcentrifuge tube, and pipet 100 ul 0.1x TE directly onto the spin column.
\n-Incubate at room temperature for 1 min, and then centrifuge for 1 min at 6000 x g (8000 rpm) to elute.
\n(May repeat with another 100 ul of 0.1x TE).
\n
Purified DNA was sent to MR DNA in stillwater Texas for Illumina sequencing of the cp-23S gene for Symbiodinium typing.
Symbiodinium community composition for individual A. cervicornis. Sampled from\u00a0Biscayne Bay Coral Nursery near Elliot Key, Florida.
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Associated Publications:
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Seibeck et al., 2006: Monitoring coral bleaching using a colour reference card
Data was processed with MR DNA's proprietary pipeline.
\n
BCO-DMO Processing Notes:
\n- modified parameter names to conform with BCO-DMO naming conventions
\n- filled all blank cells with nd