Pocillopora

DNA extraction from Pocillopora spp.

Victoria Marie Glynn Last update: 19 October 2020

Reagents

  • 15 ml Falcon tubes

  • MP Biomedicals™ Lysing Matrix A (Garnet) Bulk

  • MP Biomedicals™ 1/4 in. Ceramic Sphere

  • Bone cutters (14 cm)

  • DNA/RNA shield

  • 70% ethanol (EtOH)

  • RNAse A

  • Proteinase K (20 mg/ul)

  • UltraPure™ Phenol:Chloroform:Isoamyl Alcohol (25:24:1)

  • 3M sodium acetate (NaOAc)

  • 100% isopropanol

  • milliQ water

In advance

  • Autoclave in glass beakers enough garnet and beads for the number of samples you will be processing. Per sample, you need: 1.36g of garnet and 5 ceramic beads.

  • Autoclave and/or disinfect your bone cutters.

Day 1

  1. For each sample, grab a 15 mL Falcon tubes, and place inside: a. 8 mL of DNA/RNA shield b. 1.36g of garnet c. 5 ceramic beads.

  2. With bone cutters (14 cm gauge), cut the coral longitudinally (almost like if you were “peeling” or “pinching” the coral) and place shavings into DNA/RNA shield.

  3. Leave samples at room temperature (20-25°C) for multiple hours (until the end of the day, or 12 hours), and then store at 4°C for extraction the following day. If extraction will not occur within a week, store at -80°C.

Day 2

  1. Place 70% ethanol at 4°C.

  2. Place the 15 mL tube adapter on the vortex mixer.

  3. Vortex tubes with coral samples for 25-30 minutes on the VWR vortex mixer.

  4. Add 107 uL (20 mg/mL) of proteinase K prior to extraction.

  5. Incubate sample with proteinase K at 65°C for 4 hours.

  6. Add 10 µl RNase A. Mix by vortexing.

  7. Incubate sample with RNAse A at 37°C for 30 minutes.

  8. Spin sample at maximum speed at RT for 1 min to sediment any debris.

  9. Transfer 450uL of the supernatant to a new sterile 1.5ml tube and put on ice.

  10. In the fume hood add 450uL of 25:24:1 phenol-chloroform-isoamyl alcohol and vortex the sample briefly (only several seconds).

  11. Place the sample on ice for 1 min, and then vortex again (only several seconds).

  12. Spin the sample at maximum speed for 5 minutes at 4°C.

  13. Remove 293uL of the upper aqueous phase (No interphase!) and put in a sterile, labeled tube (Sample, Date, Initials). Write clearly.

  14. Add 30uL (1/10 volume) 3M NaOAc to the sample.

  15. Add 0.7-1 (~323 uL) volumes of room-temperature isopropanol to the DNA solution and mix well.

  16. Centrifuge the sample immediately at 10,000–15,000 x g for 15–30 min at 4°C

  17. Carefully decant the supernatant without disturbing the pellet.

  18. Wash the DNA pellet by adding 1–10 ml (depending on the size of the preparation) of COLD (4°C) 70% ethanol. This removes co-precipitated salt and replaces the isopropanol with the more volatile ethanol, making the DNA easier to redissolve. Put the lid on and #. GENTLY wash ethanol around the tube.

  19. Centrifuge at 10,000–15,000 x g for 5–15 min at 4°C. Carefully decant the ethanol supernatant and place the tube upside down on a Kimwipe.

  20. Dry the DNA pellet for ~15 mins until no liquid can be seen in the tube.

  21. f needed, leave the tube upright in the tube holder, covered by kimwipe, for an additional 5 minutes to ensure all ethanol is evaporated.

  22. Resuspend the DNA pellet in 50µl milliQ water, and then vortex the sample for no more than ~10 seconds.

  23. Store your DNA sample in a -20°C fridge.