Lab Plan for Holobiont Integration project

Protocols

Tissue extraction for molecular work
Beadbeating protocol: “Sample Prep” in K. Wong’s post
Separating symbionts from host in a tissue slurry: Protocol

DNA & RNA extraction
Protocol: Zymo-Duet

To test RNA and DNA quantity: Qubit
To test RNA quality: TapeStation
To test DNA quality: Gel Electrophoresis

Tissue extraction for physiology
Airbrushing: HIMB Protocol

Chlorophyll concentration
Following the protocol in: Wall et al 2018

Lipid content
Following Chris Wall’s protocol: Lipids
Check on these supplies:

• Chloroform ACS grade
• Methanol ACS grade
• 0.88% KCl
• UHP grade 5.0 nitrogen
• Glass fiber filters
• aluminum pans
• large and small amber vials
• cork borer and sharpener

Surface area
Wax-dipping: Following Stimson and Kinzie 1991
Method should vary with species of coral (depending on coralite structure), but single wax dip is the most effective (Veal et al 2010).
Small corallite (Porites sp., Stylophora sp.) structure - single wax dip
Acropora sp. needs double wax dip to seal deep corallites
Complex corallites (Goniastrea sp.) - single wax dip

Cell counts
“By-hand” Protocol: Symbiont Density
Flow Cytometer:
Prep cells

Total protein
Protocol: Pierce BCA

Dry biomass
Portions of the tissue slurry were dried (60 °C) to calculate dry biomass.
Protocol: ___

Ash-free dry weight
Portions of the tissue slurry were baked in a muffle furnace (4 hours at 500 °C) to calculate ash free dry weight as the difference between dry weight and the ash weight
Protocol: __
Can be done in the lipid protocol

Schedule

Week of June 3rd - June 7th: lab work planning, 4 coral fragment RNA DNA protocol testing 2 rounds, bead beating testing 1 round. Week of June 10th - 14th: Emma out of town the 11th through the 18th.
Week of June 17th - 21st: order materials, lab work planning, outside of lab work. Week of June 24th - 28th:
Week of July 1st - July 5th:
Week of July 8th - July 12th:
Week of July 15th - July 19th:
Week of July 22nd - July 26th:
Week of July 29th - August 2nd:
Week of August 5th - August 9th:
Week of August 12th - August 16th:
Week of August 19th - August 23rd:
Week of August 26th - August 30th:

Timeline: 10 samples a day (extractions, qubit, tapestation, gel) = ~40 days to do 399 samples total, ~24 days to do just the stress period samples.

Equipment Preparation

Lipid Protocol

Based on Chris Wall’s lipid protocol: link

Reusable
The following equipment is for 9 samples + 1 blank. If we want to run 18 samples at a time, the equipment will need to be doubled.

Check on the following equipment/supplies needed:

• Sonicator or sonication bath
• Heating plate
• Drying oven (60 °C)
• Muffle furnace (450 °C)
• Calipers
• Retort stands (2) and rings (10)
• Faucet-attached Vacuum Pump with Erlenmeyer Flask
• Nitrogen Filter and Drying Rig
• 60 mL separatory funnels (10)
• 2 oz mortars and pestels (10 sets)
• 20 mL Glass Pipettes for chemical dispensing (3)

Confirmed we have the following:

• Balance (0.0001 g)
• 50 mL Erlenmeyer Flasks (10)

Non-reusable
Check on the following:

• Glass test tube (1 per sample/blank)
• Pasteur Pipettes (1 per sample/blank)
• Glass fiber filters (GF/F) (1 per sample/blank)
• aluminum pans (1 per sample/blank)
• large amber vials (1 per sample/blank)
• small amber vials (1 per sample/blank)
• aluminum foil

Confirmed we have the following:

• nitrile gloves (small)
• teflon tape

Chemicals per sample

• Chloroform ACS grade - 32 mL
• Methanol ACS grade - 10 mL
• 0.88% Potassium chloride (KCl) - 16 mL
• UHP grade 5.0 Nitrogen (Order from BOC gases)

Total physiology samples = 310 If using 9 samples + 1 blank protocol:

• Glass test tubes (345 = 310 samples + 35 blanks)
• Pasteur Pipettes (345 = 310 samples + 35 blanks)
• Glass fiber filters (345 = 310 samples + 35 blanks)
• aluminum pans (345 = 310 samples + 35 blanks)
• large amber vials (345 = 310 samples + 35 blanks)
• small amber vials (345 = 310 samples + 35 blanks)
• Chloroform (11,040 mL = 345 x 32 mL)
• Methanol (3,450 mL = 345 x 10 mL)
• KCl (5,520 mL = 345 x 16 mL)

Flow Cytometer

• 96 round bottom well plates (4)
• paraformaldehyde
• adapter for microcentrifuge tubes?

Airbrushing

• 50 mL falcon tubes (310)
• filtered seawater
• 0.X µm filters (X)
• compressed air - scuba tank?

DNA RNA Extractions

• Zymo kits (order as needed)

Qubit, TapeStation, Gel

• RNA screentape (20)

Total Protein

• Pierce BCA kits (order as needed)

Wax Dipping

• dowels

Chlorophyll concentration

• acetone
• spectrophotometer
• glass plate

Dry biomass, ash-free dry weight

General - order as needed

• microcentrifuge tubes
• filter and non-filter pipettes tips