Goal: Determine amount of carbohydrates in Astrangia samples from 2021 experiment

See full protocol here from KW. Modified from this protocol from CB

Ariana and I tested out the carbohydrate assay this week. She used Mcap larval samples and I used a few of my adult Astrangia samples.

Samples used: FLD-0031, AST-1109, AST-1138, AST-1152

Important note: Symbionts were NOT removed for these samples, reading carbs for the entire holobiont

Equipment/materials

• 96-well plates
• Water bath (room temperature)
• Vortex
• Fume hood
• Pipettes and tips (P1000 and P200)
• Plate reader (can read absorbance at 485 nm)
• Glucose standards
• Sulfuric acid
• Phenol

Protocol

Preparing Standards

In this test run, we used standards prepared by KW. Info on how to prepare the standards is in his protocol

Assay

1. Take samples out of -80°C and thaw at room temperature
2. Label 5 mL tubes (10 tubes for standards + # of samples in the run)
   • add location in lab
3. When samples are thawing, make the standards and blanks as shown below:

Tube ID	Concentration (mg/mL)	Vol water (uL)	Vol 1 mM Glucose (ul)	Vol 10mM Glucose (uL)
B	0.0000	1000	0	0
1	0.00901	950	50	0
2	0.01802	900	100	0
3	0.02703	850	150	0
4	0.03604	800	200	0
5	0.05406	700	300	0
6	0.0901	500	500	0
7	0.1802	0	1000	0
8	0.3604	800	10	200
9	0.901	500	0	500

1. Vortex samples after thawed to mix well
2. Add 100 uL of coral homogenate and 900 uL DI water to pre-labeled tubes for all samples.
3. Set up a room temperature water bath in the fume hood with test tube rack (i.e. DI water in a plastic bin)
4. In the fume hood, add 25 uL of phenol to first sample
5. Add 2.5 mL sulfuric acid to sample
   • Caution: When the sulfuric acid is added, the tube will get very hot from the chemical reaction occurring. Hold the tube near the top to avoid heat.
6. Put tube in rack in the water bath to incubate
7. Repeat steps 7-9 for all samples/standards
8. When last sample has been placed in water bath, incubate all samples for 30 minutes. During this time, set up the plate layout for the 96-well plate
9. After 30 minutes, pipette 200 uL of sample/standard into 96-well plate following plate layout. Samples and standards will be run in triplicate.

Plate layout:

1. Read on plate reader at 485 nm

Calculations

1. Create standard curve with known standard concentrations and absorbance values (y = mx + b)
2. Using the resulting equation, convert sample absorbance to concentrations (mg/mL)
3. Multiply sample concentration (mg/mL) by total slurry volume (mL) and dilution factor (1000/v of sample, usually 100 mL), then divide by surface area (cm2) for resulting units: mg/cm2

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