DNA amounts for Pocillopora and Porites Mo’orea samples were really low in the previous extraction, and RNA quality for Porites was poor. We wanted to try bead homogenizing and incubating to see if un-digested proteins/sugars were inhibiting nucleic acid binding on the columns.

Using the Zymo Duet DNA/RNA Extraction Kit

Sample Prep

The starting volume in the tubes (tissue stored in ~300µl DNA/RNA Shield in -20) was a limitation last time when bead homogenizing because it is impossible to get all the liquid out of the tube once the beads are in there. I tried adding 300µl DNA/RNA Shield for 2 of the samples before bead homogenizing to see if this would work better.

Sample #	Site #	Date Collected	Type	Extraction method
45	2	2018/03/12	Pocillopora verrucosa	bead homogenize
62	2	2018/03/12	Pocillopora verrucosa	add 300µl then bead homogenize
44	2	2018/03/12	Massive Porites	bead homogenize
63	2	2018/03/12	Massive Porites	add 300µl then bead homogenize

• 300µl of DNA/RNA Shield was added to samples 62 and 63
• Beads from bead tubes were poured into the sample tubes
• All samples were homogenized by vortexing for ~30 seconds
• As much liquid was removed from the tubes as possible and transferred to new 1.5mL tubes
  Samples 44 and 63 had mucus mountains making it hard to aspirate the liquid, these are the Porites samples
• Appropriate volumes of PK Digestion Buffer and Proteinase K were added based on volumes

Sample #	Tube Volume	PK Dig Buffer Vol	ProK Vol
45	300µl	30µl	15µl
62	500µl	60µl	30µl
44	300µl	30µl	15µl
63	550µl	60µl	30µl

• Samples were vortexed, spun down, and placed in the Thermomixer at 55 degrees C for 5 hours (max time recommended in the kit) shaking at 1000rpm

DNA Extraction following this mostly

1. 10mM Tris HCl and Nuclease-free H20 were put in the incubator at 70 degrees C
2. After incubation, all samples were moved to new tubes (some beads had carried over from previous steps)
3. ~Equal volumes of DNA/RNA Lysis Buffer were added to each tube;

Sample #	Vol Lysis Buffer
45	345µl
62	590µl
44	345µl
62	640µl

1. Mixed samples by flicking and spinning down
2. 600µl of sample was gently added to Yellow DNA spin columns
3. Centrifuged columns at 16000 rcf for 30 seconds

4. Flow-through was transferred to new 1.5 (samples 44 and 45) or 5mL (samples 62 and 63) tubes labeled for RNA

5. Added 400µl DNA/RNA Prep Buffer gently to the yellow DNA spin columns
6. Centrifuged at 16,000 rcf (g) for 30 seconds
7. Discarded flow through (Zymo kit waste)
8. Added 700µl DNA/RNA Wash Buffer gently to the yellow DNA spin columns
9. Centrifuged at 16,000 rcf (g) for 30 seconds
10. Discarded flow through (Zymo kit waste)
11. Added 400µl DNA/RNA Wash Buffer genetly to the yellow DNA spin columns
12. Centrifuged at 16,000 rcf (g) for 2 minutes
13. Discarded flow through (Zymo kit waste)
14. Transferred yellow columns to new 1.5mL microcentrifuge tubes
15. Added 50µl warmed 10mM Tris HCl to each yellow DNA column by dripping slowly directly on the filer
16. Incubated at room temp for 5 minutes
17. Centrifuged at 16,000 rcf (g) for 30 seconds
18. Repeated last three steps for a final elution volume of 100µl
19. Labeled tubes, stored at 4 degrees C to quantify and gel the next day

RNA Extraction

1. Added equal volume 100% EtOH to the 1.5mL and 5mL tubes labeled for RNA containing the original yellow column flow through

Sample #	Vol EtOH
45	700µl
62	1180µl
44	700µl
62	1200µl

1. Vortexed and spin down to mix
2. Added 700µl of that liquid to the green RNA spin columns
3. Centrifuged at 16,000 rcf (g) for 30 seconds
4. Discarded flow through (Zymo kit waste)
5. Added 700µl to the green RNA spin columns (the rest from the 1.5mL RNA tubes)
6. Centrifuged at 16,000 rcf (g) for 30 seconds
7. Discarded flow through (Zymo kit waste)
8. Added 400µl DNA/RNA Wash Buffer gently to each green RNA column
9. Centrifuged at 16,000 rcf (g) for 30 seconds
10. Discarded flow through (Zymo kit waste)
11. Made DNase I treatment master mix:
    • 75µl DNA Digestion buffer x # of samples
    • 5µl DNase I x # of samples
12. Added 80µl DNase I treatment master mix directly to the filter of the green RNA columns
13. Incubated at room temp for 15 minutes
14. Added 400µl DNA/RNA Prep Buffer gently to each column
15. Centrifuged at 16,000 rcf (g) for 30 seconds
16. Discarded flow through (Zymo kit waste)
17. Added 700µl DNA/RNA Wash Buffer gently to the yellow DNA spin columns
18. Centrifuged at 16,000 rcf (g) for 30 seconds
19. Discarded flow through (Zymo kit waste)
20. Added 400µl DNA/RNA Wash Buffer genetly to the yellow DNA spin columns
21. Centrifuged at 16,000 rcf (g) for 2 minutes
22. Discarded flow through (Zymo kit waste)
23. Transferred green columns to new 1.5mL microcentrifuge tubes
24. Added 50µl warmed Nuclease free water to each green RNA column by dripping slowly directly on the filer
25. Incubated at room temp for 5 minutes
26. Centrifuged at 16,000 rcf (g) for 30 seconds
27. Repeated last three steps for a final elution volume of 100µl
28. Labeled 1.5mL tubes on ice afterwards, and aliquoted 5µl into PCR strip tubes to save for Qubit and Tape Station to avoid freeze-thaw
29. Stored all tubes in the -80

Qubit Done on March 21 2019

• Broad Range dsDNA and RNA Qubit protocol
• All samples were read twice
• RNA Qubit was done by Erin Chille

Sample	DNA Standard 1 (RFU)	DNA Standard 2 (RFU)	DNA 1 (ng/µl)	DNA 2 (ng/µl)	Average DNA	RNA Standard 1 (RFU)	RNA Standard 2 (RFU)	RNA 1 (ng/µl)	RNA 2 (ng/ul)	Average RNA
45	205	23006	29.2	28.4	28.7	406	10912	17.6	17.2	17.4
62	205	23006	30.0	29.4	29.7	406	10912	35.8	36.0	35.9
44	205	23006	5.92	5.76	5.83	406	10912	44.8	45.0	44.9
63	205	23006	22.6	22.2	22.4	406	10912	53.6	53.6	53.6

TapeStation

• Followed RNA TapeStation protocol
• TapeStation was done by Erin Chille and include some of her RNA extractions of Montipora larvae

Results:

Gel

• A 1.5% agarose gel was ran to check the integrity of the genomic DNA
• Following the PPP Lab protocol
• The first 7 samples are from the previous post, the final 4 are from this extraction
• Below the sample number is the amount of DNA in ng/µl from the Qubit