10 DNA only extractions from the first set of Porites and Pocillopora Corals from Mo’orea, 7 to try again, and 3 of Kevin’s Astrangia samples

Using the Zymo Quick-DNA Miniprep Plus kit

Sample Prep

Sample #	Type
43	Massive Porites
48	Pocillopora verrucosa
54	Massive Porites
191	Pocillopora verrucosa
164	Pocillopora verrucosa
168	Pocillopora verrucosa
244	Pocillopora verrucosa
269	Massive Porites
275	Massive Porites
280	Massive Porites
210	Massive Porites
52	Massive Porites
272	Massive Porites
306	Massive Porites
285	Massive Porites
58	Massive Porites
39	Massive Porites
H 15	Astrangia poculata
H 21	Astrangia poculata
H 27	Astrangia poculata

Mo’orea new samples

• Beads were poured into the new sample tubes. The new beads should be easier to pipette the liquid out of them as the do not get sucked up by the p20
• Samples were homogenized by vortexing for ~60 seconds for all samples
• Most of the liquid from the tubes was removed by pipetting. This was about 400µl. The tubes contained a small amount of liquid and un-homogenized tissue left, so 200µl of DNA/RNA shield was added to each tube and those were put back into the -20
• Following recommendations for samples in DNA/RNA Shield from the kit protocol, 200µl of Solid Tissue Buffer and 12µl of Proteinase K were added to each sample
• Samples were votexed, spun down, and incubated at 55 degrees C for 5 hours shaking at 600rpm

Mo’orea try again samples

• Whatever tissue was left was in ~300µl and the beads in the -20, another 100µl of DNA/RNA shield was added to make it roughly the same volume as the above samples
• Tubes were vortexed again for ~30 seconds to homogenize more
• 200µl of Blue Solid Tissue buffer and 12µl of Proteinase K were added to each sample (beads and all)
• Samples were votexed, spun down, and incubated at 55 degrees C for 5 hours shaking at 600rpm
• After digestion, as much liquid was removed as possible, all tubes still had some tissue in them. Sample 39 was very mucusy still
• Volume was about 500µl

Astrangia samples

• Samples were already lysed and stored as 500µl in the -80
• After the Mo’orea samples had digested for 5 hours, these samples were taken out to thaw
• 500µl of G-DNA binding buffer was added to each tube, vortexed, and spun down
• Extraction then followed the same way as the Mo’orea samples

Following DNA extraction went along exactly as previous post

Qubit

• Broad Range dsDNA Qubit protocol
• All samples were read twice

Sample	DNA Standard 1 (RFU)	DNA Standard 2 (RFU)	DNA 1 (ng/µl)	DNA 2 (ng/µl)	Average DNA
43	159	17132	46.8	47.4	47.1
48	159	17132	72.4	73.4	72.9
54	159	17132	106	109	107.5
191	159	17132	104	106	105
164	159	17132	72.8	75.2	74
168	159	17132	114	121	117.5
244	159	17132	87.4	88.8	88.1
269	159	17132	24.4	24.6	24.5
275	159	17132	24.8	23.4	24.1
280	159	17132	23.6	21.2	22.4
210	159	17132	10.8	10.9	10.8
52	159	17132	16.7	15.7	16.2
272	159	17132	38.8	38.6	38.7
306	159	17132	12.3	11.8	12
285	159	17132	8.76	9.56	9.16
58	159	17132	10.6	9.56	10.08
39	159	17132	26.4	25.8	26.1
H 15	159	17132	51.4	51.6	51.5
H 21	159	17132	13.1	12.8	13
H 27	159	17132	40	40.4	40.2

Gel Verification

• A 1.5% agarose gel was ran to check the integrity of the genomic DNA
• Following the PPP Lab protocol