RISG Zymo Adult Oyster Tissue DNA Extractions

Using Zymo Quick-DNA Miniprep Plus Kit

Extracting DNA from mantle tissue of adult oysters collected from various sites in Chesapeake Bay and Delaware Bay in Fall 2022.

Mantle tissue was dissected from the oysters, preserved in 100% EtOH and stored in a -80 freezer until processing. Before extractions, a 5 mg piece of tissue was cut, using the following protocol:

Mantle Tissue Prep

• Add 300 μL of DNA/RNA Shield to BeadBashing Lysis tubes
• Pull samples from -80 freezer and store on ice
• Sterilize cutting board, plastic weigh boat, scalpel blade, and forceps with 10% Bleach, Type II DI Water, and 70% EtOH
  • Sterilize all equipment between each sample
• Tare weigh boat in scale
• Pull tissue out of cryogenic tube, cut a pea-sized amount of tissue with scalpel, and transfer to weigh boat using forceps
• Weigh out 0.005 grams of tissue
  • Remove or add pieces of tissue until desired mass is reached
• Using forceps, transfer 5 mg piece of tissue to BeadBashing tube with DNA/RNA Shield
• Samples can either be processed the same day or stored at 4 degrees C for a few days before processing

Extraction Protocol

This is the general protocol I followed for DNA Extractions. I typically processed 12 samples at a time and could complete multiple extraction runs in a day.

Time to Completion: 3-4 hours (includes QC)

Homogenization

• Homogenize sample in Tissue Lyser for 2 minutes at 30 Hz
• Let samples sit for ~3 minutes, then spin down in minifuge
• Add 150 μL of Solid Tissue Buffer to each tube
• Add 10 μL of Proteinase K
• Vortex and spin down
• Put in thermomixer at 55 degrees C, shaking at 1000 rpm for 30 minutes
  • Halfway through, spin down in tabletop centrifuge at 13000 rpm for 1 minute
• After incubation, centrifuge for 1 minute at 13000 rpm in tabletop centrifuge
• Being careful not to disturb debris pellet, transfer all supernatant to new 1.5 mL tube

DNA Purification

• Make 1.5 mL tube with 10 mM Tris HCl pH 8.0 and warm to 70 degrees C in thermomixer
  • 100 μL x # of samples (plus error)
• Set up tubes for extraction
  • Yellow spin-columns and collection tubes
  • 1.5 mL tubes for final DNA sample
• Add 1 part volume (400 μL) of Genomic Binding Buffer to each 1.5 mL tube
• Vortex and spin down
• Add 800 μL of supernatant to yellow spin-column
• Centrifuge at 13000 rpm for 1 minute
  • Discard flow-through
• Transfer spin-columns to new collection tube
• Add 400 μL of DNA Pre-Wash Buffer to spin-column
• Centrifuge at 13000 rpm for 1 minute
  • Discard flow-through
• Add 700 μL of g-DNA Wash Buffer
• Centrifuge at 13000 rpm for 1 minute
  • Discard flow-through
• Add 200 μL of g-DNA Wash Buffer
• Centrifuge at 13000 rpm for 1 minute
  • Discard flow-through
• Transfer spin-column to final 1.5 mL tube
• Drip 50 μL of warmed 10 mM Tris HCl pH 8.0 directly onto filter
• Incubate at room temperature for 1 minute
• Centrifuge at max speed for 1 minute
• Again, drip 50 μL of warmed 10 mM Tris HCl pH 8.0 directly onto filter
• Incubate at room temperature for 1 minute
• Centrifuge at max speed for 1 minute
• Should have 100 μL of DNA

Quality Control Check

Qubit

• DNA concentrations are checked using the Biotium dsDNA Broad Range quantitation kit
• Final DNA concentrations (ng/μL) can be accessed here

Agarose Gel

• DNA quality is checked using an Agarose Gel, following the protocol for a Small 1% gel run
• Sample gel can be accessed here