At indicated dates, individual corals were visually assessed for bleaching. Bleaching was noted for those corals paling or entirely lacking in pigmentation.  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:

Chaos Buffer:

4.5M Guanidinium thiocyanate
2% N-lauroylsarcosine (sarcosyl)
50mM EDTA
25mM Tris-HCl, pH 7.5
0.2% antifoam A (Sigma)
0.1M b-mercaptoethanol (BME)

Binding buffer (BB):

6 M GuSCN 1 , (Guanidine Thiocyanate)
20 mM EDTA pH 8.0
10 mM Tris-HCl pH 7.5
4% Triton X-100

Protein wash buffer (PWB):

70 mL of ethanol (96%) was thoroughly mixed with 26 mL of BB (stable at 20 deg C for1 week).

Wash buffer (WB):

ethanol (60%),
50 mM NaCl
10 mM Tris-HCl pH 7.4
0.5 mM EDTA pH 8.0 (stored at -20 deg C)

-Allow coral fragments to sit covered in CHAOS buffer in 1.5 - 2.0 mL tube for days- weeks.
-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.
-Add 700 ul PWB to column, and centrifuge for 1 min at 6000 x g (8000 rpm). Discard flow-through.
-Repeat 2x (3 washes total).
-Add 700 ul WB to column and centrifuge for 1 min at 20,000 x g (14,000 rpm). Discard flow-through.
-Repeat wash and centrifuge for 3 min at 20,000 x g (14,000 rpm) to dry the DNeasy membrane.
-Discard flow-through and collection tube.
-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.
-Incubate at room temperature for 1 min, and then centrifuge for 1 min at 6000 x g (8000 rpm) to elute.
(May repeat with another 100 ul of 0.1x TE).

Purified DNA was sent to MR DNA in stillwater Texas for Illumina sequencing of the cp-23S gene for Symbiodinium typing.