Zymo DNA/RNA Extractions of Larval Oyster Samples

Using Zymo Quick-DNA/RNA Miniprep Plus Kit

Extracting DNA and RNA from larval oyster samples from multi-stressor exposure experiment.

Larvae were collected onto Pluri filters, preserved with liquid nitrogen, stored in Whirlpaks and held in a -80 freezer until processing.

This is the general protocol I followed for DNA/RNA Extractions. I typically processed 10-12 samples at a time.

Time to Completion: 8-10 hours (includes QC)

Homogenization

• In 50 mL falcon tubes, add 1000 μL of DNA/RNA Shield (can be prepped the night before)
• Turn on Incubator Genie and set to 55 degrees C, rocking speed 35
• Pull Proteinase K out of -20 freezer to warm to room temperature
• Sterilize scalpel blade with 100% EtOH, Type II DI Water, RNAseZap, and RNAse-free Water
  • Repeat the sterilization between each sample
• Take samples out of -80 freezer one at a time
• Place filter on open falcon tube, and cut a cross section through the filter then cut the perimeter of the filter
  • Make sure all pieces of the filter fall into the falcon tube and are submerged in DNA/RNA Shield
• Add 100 μL of PK Digestion Buffer to each tube
• Add 50 μL Proteinase K to each tube and vortex to mix
• Incubate tubes in Incubator Genie at 55 degrees C, rocking speed 35 for 2.5 hours
  • Vortex the tubes every 30 minutes
• During incubation period:
  • Prep and label all tubes for extraction protocol (for each tube type, multiply by number of samples being processed)
    • 1.5 mL tubes for digested sample
    • 5 mL tubes for intermediate DNA steps
    • 5 mL tubes for intermediate RNA steps
    • Yellow spin-column and collection tubes for DNA purification
    • Green or white spin-column and collection tubes for RNA purification (spin-column color will depend on kit)
    • 1.5 mL tubes for final DNA sample
    • 1.5 mL tubes for final RNA sample
  • Prep 10 mM Tris HCl pH 8.0 and RNAse-free Water for thermomixer
    • For each, 100 μL x # of samples (plus error) in 1.5 mL tubes
    • Put in thermomixer set to 70 degrees C
  • Make DNAse reaction mix:
    • DNA Digestion Buffer: 75 μL x # of samples
    • DNAse I: 5 μL x # of samples
• After incubation, spin down falcon tubes in large centrifuge at max speed for 2 minutes
• Transfer all liquid, minus filter pieces, to 1.5 mL tubes
• Centrifuge tubes in tabletop centrifuge at max speed for 2 minutes
• Being careful not to disturb debris pellet, transfer all liquid to 5 mL tube for DNA
  • Save debris pellet in labeled 1.5 mL tube and store in -20 freezer (can check for incomplete digestion later on if necessary)
• Add equal volume (1000 μL) of DNA/RNA Lysis Buffer to each 5 mL tube for DNA
  • Finger flick to mix
• Transfer 700 μL of supernatant to yellow spin column
• Centrifuge at 16000 rpm for 30 seconds
• Transfer flow-through to 5 mL tube for RNA
• Repeat until all supernatant is filtered (3 times total)
• Pull Biotium dsDNA and RNA kits out of fridge/freezer and put in drawer (light sensitive) to warm to room temperature

DNA Purification

• Add 400 μL of DNA/RNA Prep Buffer to yellow spin-column
• Centrifuge at 16000 rpm for 30 seconds
  • discard flow-through
• Add 700 μL of DNA/RNA Wash Buffer
• Centrifuge at 16000 rpm for 30 seconds
  • discard flow-through
• Add 400 μL of DNA/RNA Wash Buffer
• Centrifuge at 16000 rpm for 2 minutes
  • discard flow-through
• Transfer spin-column to final 1.5 mL tube
• Carefully drip 50 μL of warmed 10 mM Tris HCl pH 8.0 directly onto filter
• Incubate at room temperature for 5 minutes
• Centrifuge at 16000 rpm for 30 seconds
  • KEEP FLOW-THROUGH
• Again, carefully drip 50 μL of warmed 10 mM Tris HCl pH 8.0 directly onto filter
• Incubate at room temperature for 5 minutes
• Centrifuge at 16000 rpm for 30 seconds
• Should have 100 μL of DNA

RNA Purification

• Add equal volume (2000 μL) 100% EtOH to each 5 mL tube for RNA
• Transfer 700 μL of supernatant to white spin-column
• Centrifuge at 16000 rpm for 30 seconds
  • Discard flow-through
• Repeat until all supernatant is filtered (6 times total)
• Add 400 μL of DNA/RNA Wash Buffer to white spin-column
• Centrifuge at 16000 rpm for 30 seconds
  • discard flow-through
• Add 80 μL of DNAse reaction mix to each column (I add directly onto filter)
• Incubate at room temperature for 30 minutes
• Add 400 μL of DNA/RNA Prep Buffer to white spin-column
• Centrifuge at 16000 rpm for 30 seconds
  • discard flow-through
• Add 700 μL of DNA/RNA Wash Buffer
• Centrifuge at 16000 rpm for 30 seconds
  • discard flow-through
• Add 400 μL of DNA/RNA Wash Buffer
• Centrifuge at 16000 rpm for 2 minutes
  • discard flow-through
• Transfer spin-column to final 1.5 mL tube
• Carefully drip 50 μL of warmed RNAse-free Water directly onto filter
• Incubate at room temperature for 5 minutes
• Centrifuge at 16000 rpm for 30 seconds
  • KEEP FLOW-THROUGH
• Again, carefully drip 50 μL of warmed RNAse-free Water directly onto filter
• Incubate at room temperature for 5 minutes
• Centrifuge at 16000 rpm for 30 seconds
• Should have 100 μL of RNA

Quality Control Check

Qubit

• DNA and RNA concentrations are checked using the Biotium dsDNA Broad Range and RNA Broad Range quantitation kits
• Final DNA and RNA 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

TapeStation

• RNA quality is checked using the RNA ScreenTape Assay
• Sample TapeStation report can be accessed here