TITLE: Coral Carbohydrate Assay for 96-well plates
AUTHORS: Colleen Bove, Justin Baumann

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><ol style = "list-style-type: decimal;"></ol></div><div class = "text-block">1. Masuko T, Minami A, Iwasaki N, Majima T, Nishimura S, Lee YC. Carbohydrate analysis by a phenol-sulfuric acid method in microplate format. Anal Biochem. 2005 Apr 1;339(1):69-72. doi: 10.1016/j.ab.2004.12.001. PMID: 15766712.</div></div>

[STEPS]
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Pull desired coral slurries from the -80 freezer to thaw at room temperature
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Collect test tubes and label (10 for the standard + the number of samples running)
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Make the standards and blank as shown below Tube IDConcentration (mg/mL)Vol water (uL)Vol 1 mM GlcoseVol 10mM GlucoseB0.000010000010.0090195050020.01802900100030.02703850150040.03604800200050.05406700300060.0901500500070.180201000080.36048001020090.9015000500
Tube IDConcentration (mg/mL)Vol water (uL)Vol 1 mM GlcoseVol 10mM GlucoseB0.000010000010.0090195050020.01802900100030.02703850150040.03604800200050.05406700300060.0901500500070.180201000080.36048001020090.9015000500
?. [Carbohydrate Assay]
Vortex samples after thawing (samples were previously broken up via bead beating with glass beads)
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Set up a room temperature water bath in the fume hood with test tube rack (DI water in a plastic bin is fine)
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Add 25 uL of phenol to first sample
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Vortex (in the hood) for 3 seconds
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Immediately add 2.5 mL sulphuric acid to the sample
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Incubate the sample at room temperature for 1 minute
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Transfer sample to water bath
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Repeat steps 6-10 for all tubes
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When the last sample is placed in the water bath, incubate all samples for 30 minutes
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Pipette 200 uL of all standards and samples into the bottom of the wells in a 96-well plate (see the two different plate layouts for either sets of 3 or 4 per sample)Plate layout for 3 sample replicates123456789101112ABBB888S7S7S7S15S15S15B111999S8S8S8S16S16S16C222S1S1S1S9S9S9S17S17S17D333S2S2S2S10S10S10S18S18S18E444S3S3S3S11S11S11S19S19S19F555S4S4S4S12S12S12S20S20S20G666S5S5S5S13S13S13S21S21S21H777S6S6S6S14S14S14S22S22S22Plate layout for 4 sample replicates123456789101112ABBB888S4S6S8S10S12S14B111999S4S6S8S10S12S14C222S1S2S3S4S6S8S10S12S14D333S1S2S3S4S6S8S10S12S14E444S1S2S3S5S7S9S11S13S15F555S1S2S3S5S7S9S11S13S15G666S5S7S9S11S13S15H777&nbsp;&nbsp;&nbsp;S5S7S9S11S13S15
Plate layout for 3 sample replicates123456789101112ABBB888S7S7S7S15S15S15B111999S8S8S8S16S16S16C222S1S1S1S9S9S9S17S17S17D333S2S2S2S10S10S10S18S18S18E444S3S3S3S11S11S11S19S19S19F555S4S4S4S12S12S12S20S20S20G666S5S5S5S13S13S13S21S21S21H777S6S6S6S14S14S14S22S22S22Plate layout for 4 sample replicates123456789101112ABBB888S4S6S8S10S12S14B111999S4S6S8S10S12S14C222S1S2S3S4S6S8S10S12S14D333S1S2S3S4S6S8S10S12S14E444S1S2S3S5S7S9S11S13S15F555S1S2S3S5S7S9S11S13S15G666S5S7S9S11S13S15H777&nbsp;&nbsp;&nbsp;S5S7S9S11S13S15
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Cover plate and then read on spectrophotometer at 485 nm and then calculate sample concentrations following method listed under 'Materials'
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Add 100 uL of coral slurry and 900 uL milliQ water to pre-labelled test tube for all samples*You many need to concentrate or dilute your samples more depending on how they were collected.