In this Extraction I used: Wizard Genomic DNA Purification Kit, and my Protocol.

Goal: Extract genomic DNA from Stylophora pistillata shallow and mesophotic planulae

Steps

• For each sample to be processed, add 120 uL of a 0.5M EDTA solution (pH 8.0) to 500 uL of Nuclei Lysis Solution in a centrifuge tube. Chill on ice. #Note: the solution will turn cloudy when chilled.
• Add 5-10 planulae to a 1.5 mL microcentrifuge tube.
• Add 600 uL of EDTA/Nuclei Lysis Solution from the first step to the tube.
• Add 17.5 uL of 20mg/ml Proteinase K.
• Incubate 3 hours at 55°C (with shaking); vortex the sample once per hour, make sure that the planulae is completely digested.
• To the room temperature sample, add 200 uL of Protein Precipitation Solution and vortex at high speed for 20 seconds. Chill sample on ice for 5 minutes.
• Centrifuge for 4 minutes at 13,000-16,000g. The precipitated protein will form a tight white pellet.
• Carefully remove the supernatant containing the DNA (leaving the protein pellet behind) and transfer it to a clean 1.5 ml microcentrifuge tube containing 600 ul of room temperature isopropanol. #Note: Some supernatant may remain in the original tube containing the protein pellet. Leave this residual liquid in the tube to avoid contaminating the DNA solution with the precipitated protein.
• gently mix the solution by inverting the tube until the white thread-like strands of DNA form a visible mass.
• Centrifuge for 1 minute at 13,000-16,000g at room temperature. The DNA will be visible as a small white pellet. Carefully decant the supernatant.
• Add 600 ul of room temperature 70% ethanol, and gently invert the tube several times to wash the DNA. Centrifuge for 1 minute at 13,000-16,000g at room temperature.
• Carefully aspirate the ethanol using either a drawn Pasteur pipette or a sequencing pipette tip. The DNA pellet is very loose at this point, and care must be used to avoid aspirating the pellet into the pipette.
• Invert the tube on clean absorbent paper, and air-dry the pellet for 10-15 minutes.
• Add 50 ul of nuclease-free water and rehydrate the DNA by incubating at 65°C for 1 hour. Periodically mix the solution by gently tapping the tube. Alternatively, rehydrate the DNA by incubating the solution overnight at room temperature or at 4°C.
• Store the DNA at 2-8°C and proceed to measure the concentration.