Using the Zymo Quick-DNA/RNA Miniprep Plus Kit on adult coral fragments. This protocol is based off of the protocol of Zoe Dellaert with some adjustments by me.

Reagents preparation (first time opening the kit)

1. Add 96 mL 100% ethanol (104 mL 95% ethanol) to the 24 mL DNA/RNA Wash Buffer concentrate before use. DNA/RNA Wash Buffer included with D7003T (Mini Prep Plus Kit) is supplied ready-to-use and does not require the addition of ethanol prior to use. Check kit contents and instructions to confirm prep steps.
2. Reconstitute the lyophilized (freeze-dried) DNase I as indicated on the vial prior to use. Mix by inversion. Store frozen aliquots.
3. Reconstitute the lyophilized (freeze-dried) Proteinase K as indicated on the vial prior to use. For 20g Proteinase K, add 1040 uL Proteinase K Storage Buffer. For 5 mg Proteinase K, add 260 uL Proteinase K Storage Buffer. Mix by vortexing. Check kit contents and instructions to confirm prep steps. Store tube at -20 ºC, no need to aliquot.

Sterilizing working area to maintain an RNAse-free environment

Clean bench with clean paper towels (spray solution, wipe down) in the following order: 10% bleach solution DI water 70% ethanol RNAse cleaner (spray bottle) Clean pipettes, tip boxes, and the controls on the heating block and centrifuge by squirting 70% ethanol on a paper towel and wiping them down. Repeat with RNAse cleaner. Wipe down gloves with 70% ethanol and RNAse cleaner. Do not spray solutions directly on the equipment.

Sample preparation:

Adult Fragment Sample stored in Zymo DNA/RNA Shield

• This protocol utilizes coral clippings stored in 1mL of Zymo DNA/RNA shield and kept at -80 ºC. Thaw samples to room temperature on benchtop. On a clean work surface (see above), stand up the tubes in numerical order and photograph to record qualitative qualities such as buffer color and amount of tissue in tube.
• Take 300 uL of the DNA/RNA shield from the sample tube into a clean 1.5 mL tube and performing a Proteinase K digestion in a new tube (described below).

  Notes on sample preparation

• If the DNA/RNA shield looks very dark (ex: this occurs a lot with highly pigmented species such as Porites spp.), only take 150 or 100 uL of the DNA/RNA shield from the sample tube into the new tube and dilute with 150 uL or 200 uL of clean DNA/RNA shield (provided in kit).

• If the extraction is not yielding enough DNA or RNA and the DNA/RNA shield looks very light-colored, it could be that the lytic properties of the DNA/RNA shield did not break up the tissue enough to yield enough DNA and RNA from the buffer alone. In this case, you could bead beat this sample using the protocols described by other lab members Emma L. Strand, Dr. Kevin H. Wong, and Kristina Terpis. This bead beating step could be done by adding beads to the original sample tube, or by transferring an aliquot (300 uL) of the DNA/RNA shield from the sample tube along with a clipping of the tissue fragment into a new bead tube. Add 500-700 uL of clean DNA/RNA shield to make up 800 uL - 1 mL for bead-beating and bead beat for 1-2 minutes at maximum speed. Then, centrifuge briefly and take an aliquot of 300 uL from this homogenized mixture into a clean 1.5 mL tube. Proceed with Proteinase K digestion.

Adult Fragment Sample flash frozen, not stored in Zymo DNA/RNA Shield

• With sterilized clippers (10% bleach, DEPC water, 70% ethanol, air-dried), cut small (5mm x 5mm) fragments from the frozen sample into a clean 1.5 mL screw-cap tube with 1000 uL DNA/RNA shield and 0.25 mL of 0.5mm glass beads, Work on ice (better if dry ice). Sterilize clippers between each sample.
  Note: for species with a lot of mucus, like Montipora capitata, I used 1300 uL of DNA/RNA shield to try to dilute as much as possible the mucus and avoid getting it into the extraction column.
• Bead beat for 1-2 minutes on the vortex at max speed.
• Briefly spin down and remove 400 uL of supernatant into a clean tube. Note: for Porites compressa the DNA/RNA shield looked very dark, so I only took 150 of the DNA/RNA shield from the sample tube into the new tube and diluted with 250 uL of clean DNA/RNA shield.
• Spin for 3-4 mins at 9,000 rcf (a little pellet forms at the bottom of the tubes)
• Put original samples and bead tubes back in -80 ºC freezer.
• Remove 300 uL of the supernatant into a new tube and continue on with the protocol below (from the Pro K step) as written.

Proteinase K Digestion

• Add: 30 µl of PK digestion buffer to each 300 uL sample tube (1:10 ratio of PK Digestion Buffer:Sample)
• Add: 15 µl Proteinase K to each (1:2 ratio of Proteinase K:PK Digestion Buffer).
• Invert 3x to mix.
• Vortex and spin down.
• Place original sample tubes back in the freezer box (in -80 ºC) that they came from. Always keep these as backup.

DNA Extraction

1. Set up yellow DNA spin columns and collection tubes, label appropriately
2. Warm elution liquids to 70°C (10mM Tris HCl pH. 8.0 and RNase free water)
3. Add equal volume (345 µl) DNA/RNA lysis buffer to each sample tube
4. Finger flick to mix tubes
5. Add 700 µl (total volume) of sample gently to the yellow DNA spin column
6. Centrifuge at 16,000 rcf (g) for 30 seconds
7. Important Save the flow through from this step: transfer to a new 1.5mL tube labeled for RNA
8. Add 400µl DNA/RNA Prep Buffer gently to the yellow DNA spin columns
9. Centrifuge at 16,000 rcf (g) for 30 seconds
10. Discard flow through (Zymo kit waste)
11. Add 700 µl DNA/RNA Wash Buffer gently to the yellow DNA spin columns
12. Centrifuge at 16,000 rcf (g) for 30 seconds
13. Discard flow through (Zymo kit waste)
14. Add 400 µl DNA/RNA Wash Buffer genetly to the yellow DNA spin columns
15. Centrifuge at 16,000 rcf (g) for 2 minutes
16. Discard flow through (Zymo kit waste)
17. Transfer yellow columns to new 1.5mL microcentrifuge tubes
18. Add 50 µl warmed 10 mM Tris HCl to each yellow DNA column by dripping slowly directly on the filter
19. Incubate at room temp for 5 minutes
20. Centrifuge at 10,000 rcf (g) for 30 seconds - higher than 10,000 rcf the tube caps will detach
21. Repeat steps 18-20 for a final elution volume of 100 µl
22. Label tubes, store at 4°C if quantifying the same day or the next (Nanodrop), if waiting longer store in -20°C

RNA Extraction

Can do concurrently with DNA Extraction after DNA Extraction Step 7

1. Add equal volume (700 µl) 100% EtOH to the 1.5mL tubes labeled for RNA containing the original yellow column flow through
2. Vortex and spin down to mix
3. Add 700 µl of that liquid to the green RNA spin columns
4. Centrifuge at 16,000 rcf (g) for 30 seconds
5. Discard flow through (Zymo kit waste)
6. Add 700 µl to the green RNA spin columns (the rest from the 1.5mL RNA tubes)
7. Centrifuge at 16,000 rcf (g) for 30 seconds
   • Get DNase I from freezer
8. Discard flow through (Zymo kit waste)
9. Add 400 µl DNA/RNA Wash Buffer gently to each green RNA column
10. Centrifuge at 16,000 rcf (g) for 30 seconds
11. Discard flow through (Zymo kit waste)
12. Make DNase I treatment master mix:
    • 75µl DNA Digestion buffer x # of samples
    • 5µl DNase I x # of samples
13. Add 80 µl DNase I treatment master mix directly to the filter of the green RNA columns
14. Incubate at room temp for 15 minutes
15. Add 400 µl DNA/RNA Prep Buffer gently to each column
16. Centrifuge at 16,000 rcf (g) for 30 seconds
17. Discard flow through (Zymo kit waste)
18. Add 700 µl DNA/RNA Wash Buffer gently to the green RNA spin columns
19. Centrifuge at 16,000 rcf (g) for 30 seconds
20. Discard flow through (Zymo kit waste)
21. Add 400 µl DNA/RNA Wash Buffer genetly to the green RNA spin columns
22. Centrifuge at 16,000 rcf (g) for 2 minutes
23. Discard flow through (Zymo kit waste)
24. Transfer green columns to new 1.5mL microcentrifuge tubes
25. Add 50µl warmed DNase/RNase free water to each green RNA column by dripping slowly directly on the filer
26. Incubate at room temp for 5 minutes
27. Centrifuge at 10,000 rcf (g) for 30 seconds - higher than 10,000 rcf the tube caps will detach
28. Repeat steps 25-27 for a final elution volume of 100µl
29. Label 1.5mL tubes on ice afterwards, and aliquot 5µl into PCR strip tubes to save for Nanodrop and Tape Station to avoid freeze-thaw of your stock sample
30. Store all tubes in the -80°C

DNA and RNA quantity and quality

• Follow Broad Range dsDNA and RNA Qubit protocol to analyze sample quantity. Read standards once and record values, read all samples twice.
• Run DNA and RNA samples on an agarose gel
• If RNA quantity is sufficient (for sequencing you need to have a total of 500 ng per sample), determine RNA quality using the Tape Station. “Good” RNA should have a RIN above 8.0 and form two distinct peaks at the 18S and 28S locations.