RISG DNA Sonication

Sonication of oyster DNA samples for shearing to 300 bp

DNA extractions were previously performed on both adult and larval oyster samples.

This is the general protocol I followed for DNA Sonication. I typically processed 16 samples at a time and could complete multiple sonication runs in a day.

Sonication

  • Samples were sonicated following the QSonica Protocol
  • Prep samples in QSonica tubes
    • Aliquot 500 ng of DNA sample into tube (calculated based on Qubit concentrations after DNA extractions)
    • Add 10 mM Tris HCl pH 8.0 to achieve a total volume of 100 μL
  • Vortex and spin down
  • Sonicate samples for 5 minutes
    • Pulse setting at 15 seconds on and 15 seconds off
    • Amplitude = 35%
    • Pause program and spin down tubes every 2 minutes

1.8X Bead Clean

After sonicating, perform 1.8X bead clean with KAPA Pure Beads

  • Make 80% Ethanol (make fresh daily)
    • For a total batch of 20 mL, add 16 mL of 200 proof EtOH and 4 mL Type II DI Water
  • Take KAPA Pure Beads out of 4 degrees C fridge and keep in drawer (light sensitive) to warm to room temperature for 30 minutes
  • Add 180 μL of KAPA Pure Beads to each sample and pipette to mix
  • Incubate at room temperature on shaker for 15 minutes
  • Place tubes on magnet and remove and discard supernatant (~277 μL) when clear
  • Add 200 μL of 80% EtOH to each tube while still on magnet - don’t disturb beads collected on magnet
  • Remove and discard 197 μL of clear supernatant
  • Add 190 μL of 80% EtOH to each tube while still on magnet - don’t disturb beads
  • Remove and discard all (200 μL) clear supernatant
  • Remove any droplets collected on sides of tube with a p20 pipette tip
  • Don’t let beads sit dry for more than 2 minutes, this can result in large losses of DNA
  • Take tubes off of magnet, and resuspend the beads in 30 μL room temperature 10 mM Tris HCl pH 8.0
  • Incubate on shaker for 5 minutes
  • Make a set of final PCR strip tubes
  • Place samples on magnet, transfer clear supernatant to final PCR strip tubes

Quality Control Check

Qubit

  • Concentrations for a subset of sonicated DNA samples are checked using the Biotium dsDNA Broad Range quantitation kit

TapeStation

  • Shearing accuracy is checked for a subset of samples using the D1000 ScreenTape Assay
  • Sample TapeStation report can be accessed here
Written on February 1, 2024