Using the Zymo Quick-DNA/RNA Microprep Plus Kit on coral spat settled on plugs. This protocol is based off of the protocol of Maggie Schedl and the kit protocol with 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: frozen spat settled on plugs

Sample Scraping and Homogenizing

• Set up a cryo tube per each sample with 500ul DNA/RNA Shield in each and 0.25 mL of 0.5mm glass beads
• Using a sterile razor blade, scrape the desired amount of spat from the frozen plug, work on dry ice
• Scrape the spat off onto the cry tupe with the shield
• Close the lid of the tube and invert a few times to submerge the tissue
• If the liquid is colored there is a reasonable amount of tissue for extraction
• Bead beat the tube for 1 minute on the vortex at max speed.
• Briefly spin down and remove 350 uL of supernatant into a clean tube.
• Spin for 1 minute 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

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.

DNA Extraction

1. Set up yellow DNA spin columns and collection tubes, label appropriately
2. Add equal volume (345 µl) DNA/RNA lysis buffer to each sample tube
3. Finger flick to mix tubes
4. Add 700 µl (total volume) of sample gently to the yellow DNA spin column
5. Centrifuge at 16,000 rcf (g) for 30 seconds
6. Important Save the flow through from this step: transfer to a new 1.5mL tube labeled for RNA
7. Add 400µl DNA/RNA Prep Buffer gently to the yellow DNA spin columns
8. Centrifuge at 16,000 rcf (g) for 30 seconds
9. Discard flow through (Zymo kit waste)
10. Add 700 µl DNA/RNA Wash Buffer gently to the yellow DNA spin columns
11. Centrifuge at 16,000 rcf (g) for 30 seconds
12. Discard flow through (Zymo kit waste)
13. Add 400 µl DNA/RNA Wash Buffer genetly to the yellow DNA spin columns
14. Centrifuge at 16,000 rcf (g) for 2 minutes
15. Discard flow through (Zymo kit waste)
16. Transfer yellow columns to new 1.5mL microcentrifuge tubes
17. Add 15 µl of DNase/RNase free water to each yellow DNA column by dripping slowly directly on the filter
18. Incubate at room temp for 1 minute
19. Centrifuge at 10,000 rcf (g) for 30 seconds - higher than 10,000 rcf the tube caps will detach
20. Label tubes, store at 4°C if quantifying the same day or the next (Qbit), 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:
    • 35µl DNA Digestion buffer x # of samples
    • 5µl DNase I x # of samples
13. Add 40 µl DNase I treatment master mix directly to the filter of the green RNA columns
14. Incubate at room temp for MAX 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 15µl of DNase/RNase free water to each green RNA column by dripping slowly directly on the filer
26. Incubate at room temp for 1 minute
27. Centrifuge at 10,000 rcf (g) for 30 seconds - higher than 10,000 rcf the tube caps will detach
28. Label 1.5mL tubes (if not already done), and aliquot 1µl into a Qbit tube for quantification and 3.5µl into a 0.5 tube for gel, to avoid freeze-thaw of your stock sample
29. Store extracted RNA in the -80°C

DNA and RNA quantity and quality

• Follow Broad Range dsDNA and high sensitivity RNA Qubit protocols to analyze sample quantity. Read standards once and record values, read all samples twice.
• Run DNA and RNA samples on an agarose gel