CADO Zymo DNA/RNA Extractions of Small Juvenile Oyster Samples

Zymo DNA/RNA Extractions of Juvenile Oyster (< 4 mm) Samples

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

Juveniles were collected individually in 1.5 mL tubes, preserved with liquid nitrogen, and held in a -80 freezer until processing.

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

Time to Completion: 6-8 hours (includes QC)

Homogenization

  • 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
  • In 1.5 mL tubes, add 300 μL of DNA/RNA Shield
  • Pull samples out of -80 freezer and put on ice
  • Sterilize forceps with 100% EtOH, Type II DI Water, RNAse Zap, and RNAse-free Water
    • Sterilize forceps between each sample
  • Using forceps, transfer juvenile to 1.5 mL tube with DNA/RNA Shield
  • Add 30 μL PK Digestion Buffer to each tube
  • Add 15 μL Proteinase K
  • Vortex for 30 seconds and spin down
  • Incubate in Incubator Genie at 55 degrees C for 2 hours
    • Every 30 minutes, vortex samples for 30 seconds and spin down
  • 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 intermediate DNA
      • 1.5 mL tubes for intermediate RNA
      • 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, 50 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 tubes at max speed for 2 minutes in tabletop centrifuge
  • Without disturbing the debris pellet, gently transfer all supernatant (~350 μL) to new 1.5 mL tube for DNA
    • Save debris pellet and store in -20 freezer (can check for incomplete digestion later on if necessary)
  • Add equal volume (300 μL) of DNA/RNA Lysis Buffer to each tube
    • Finger flick to mix
  • Transfer all supernatant (~600 μL) to yellow spin-column
  • Centrigue at 16000 rpm for 30 seconds
  • Transfer flow-through to new 1.5 mL tube for RNA

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
  • Pipette the 50 μL out of the 1.5 mL tube and drip directly onto the filter again
  • Incubate at room temperature for 5 minutes
  • Centrifuge at 16000 rpm for 30 seconds
  • Should have 50 μL of DNA

RNA Purification

  • Add equal volume (600 μL) 100% EtOH to each 1.5 mL tube for RNA
  • Transfer 600 μL of supernatant to white spin-column
  • Centrifuge at 16000 rpm for 30 seconds
    • Discard flow-through
  • Repeat until all supernatant is filtered (2 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
  • Pipette the 50 μL out of the 1.5 mL tube and drip directly onto the filter again
  • Incubate at room temperature for 5 minutes
  • Centrifuge at 16000 rpm for 30 seconds
  • Should have 50 μL of RNA

Quality Control Check

Qubit

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

Written on February 1, 2024