Pocillopora
===========

**DNA extraction from Pocillopora spp.**

Victoria Marie Glynn
Last update: 19 October 2020 

Reagents
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*  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
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*  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
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#. 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.
#. 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. 
#. 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
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#. Place 70% ethanol at 4°C.
#. Place the 15 mL tube adapter on the vortex mixer. 
#. Vortex tubes with coral samples for 25-30 minutes on the VWR vortex mixer. 
#. Add 107 uL (20 mg/mL) of proteinase K prior to extraction. 
#. Incubate sample with proteinase K at 65°C for 4 hours.
#. Add 10 µl RNase A. Mix by vortexing. 
#. Incubate sample with RNAse A at 37°C for 30 minutes.
#. Spin sample at maximum speed at RT for 1 min to sediment any debris.
#. Transfer 450uL of the supernatant to a new sterile 1.5ml tube and put on ice.
#. In the fume hood add 450uL of 25:24:1 phenol-chloroform-isoamyl alcohol and vortex the sample briefly (only several seconds). 
#. Place the sample on ice for 1 min, and then vortex again (only several seconds). 
#. Spin the sample at maximum speed for 5 minutes at 4°C.
#. Remove 293uL of the upper aqueous phase (No interphase!) and put in a sterile, labeled tube (Sample, Date, Initials). Write clearly.
#. Add 30uL (1/10 volume) 3M NaOAc to the sample.
#. Add 0.7-1 (~323 uL) volumes of room-temperature isopropanol to the DNA solution and mix well.
#. Centrifuge the sample immediately at 10,000–15,000 x g for 15–30 min at 4°C
#. Carefully decant the supernatant without disturbing the pellet.
#. 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.
#. 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.
#. Dry the DNA pellet for ~15 mins until no liquid can be seen in the tube. 
#. f needed, leave the tube upright in the tube holder, covered by kimwipe, for an additional 5 minutes to ensure all ethanol is evaporated.
#. Resuspend the DNA pellet in 50µl milliQ water, and then vortex the sample for no more than ~10 seconds.
#. Store your DNA sample in a -20°C fridge.