DNA Extraction Protocol For the Zymo Quick-DNA Miniprep Plus kit On Corals Stored in DNA/RNA Shield and Frozen at -20

Homogenization and Incubation

• Thaw the samples on ice
• Sample tubes should contain tissue piece and ~500ul of DNA/RNA Shield
• Add 150ul of ultra pure molecular grade water to each 1.5 ml tube
• Add 150ul of Solid Tissue Buffer (blue) to each 1.5mL tube
• Add 10ul proteinase K to each 1.5mL tube
• Vortex 1.5mL tubes and spin down briefly
• Start the thermomixer and set to 55 degrees C
• Place samples in the thermomixer and set the shaking speed to 600rpm
• Incubate samples in the thermomixer for 5 hours (time can be increased or decreased: troubleshoot this step for your samples/species)

DNA Extraction

• Label final 1.5mL tubes per sample
• Warm DNA Elution Buffer or 10mM Tris HCl to 70 degrees C in the thermomixer (you’ll need 100ul per sample)
• After incubation, centrifuge tubes at 12,000 rcf for 1 minute to pellet any debris and beads you may have sucked up
• Remove supernatant into new 1.5mL tubes (this should be about 200-300ul). Discard the tube from incubation
• Add equal volume (200-300µl) Genomic Binding Buffer to each 1.5mL tube
• Vortex each 1.5mL tube to mix
• Spin down each 1.5mL tube in the minifuge to collect the liquid
• Set up one spin column and collection tube per sample
• Add 400ul of sample (not all the volume) to the spin column
• Centrifuge spin columns at 12,000 rcf for 1 minute
• Discard the collection tube and use a new one
• Add the remainder of the liquid from the 1.5mL tubes to the spin columns
• Centrifuge spin columns at 12,000 rcf for 1 minute
• Discard the collection tube and use a new one
• Add 400µl DNA Pre-Wash Buffer to each spin column
• Centrifuge columns at 12,000 rcf for 1 minute
• Pour off flow through into a liquid waste beaker
• Add 700µl G-DNA Wash Buffer to spin columns
• Centrifuge columns at 12,000 rcf for 1 minute
• Pour off flow through into a liquid waste beaker
• Add 200µl G-DNA Wash Buffer to columns
• Centrifuge columns at 12,000 rcf for 1 minute
• Pour off flow through into a liquid waste beaker and discard the collection tube
• Transfer spin columns to the final labeled 1.5mL tubes
• Add 50ul warmed 70 degrees C DNA Elution Buffer or 10mM Tris-HCl directly to the column filter
• Incubate the columns at room temp for 5 minutes on the bench
• Centrifuge columns at 12,000 rcf for 1 minute
• Again add 50ul warmed 70 degrees C DNA Elution Buffer or 10mM Tris-HCl directly to the column filter
• Incubate the columns at room temp for 5 minutes on the bench
• Centrifuge columns at 12,000 rcf for 1 minute
• Aliquot 8ul of the DNA into strip tubes for QC analysis
• Freeze the 1.5mL tubes at -20
• Keep the strip tube aliquots on ice if doing QC the same day, or freeze at -20 if doing another day

QC

Qubit: Broad Range DNA

• Follow Qubit protocol
• Read samples twice and average readings
• Make sure to record standards

DNA Gel

• Follow Gel Protocol
• Suggestions are to:
  • Make a 1% gel and run for 1hr at 100V
  • Use 3ul 1kb plus DNA ladder
  • Use gel green