18 DNA only extractions from Porites and Pocillopora Corals from Mo’orea

Using the Zymo Quick-DNA Miniprep Plus kit

Sample Prep

Sample #	Type
5	Massive Porites
8	Massive Porites
49	Pocillopora verrucosa
50	Pocillopora verrucosa
67	Massive Porites
70	Massive Porites
192	Pocillopora verrucosa
198	Massive Porites
220	Pocillopora verrucosa
246	Pocillopora verrucosa
338	Massive Porites
364	Pocillopora verrucosa
172	Pocillopora verrucosa
176	Pocillopora verrucosa
186	Pocillopora verrucosa
384	Pocillopora verrucosa
405	Massive Porites
424	Massive Porites

• New Beads were poured into 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 ~30 seconds for all samples
• Most of the liquid from the tubes was removed by pipetting. This was about 300µl. 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, 150µl of Solid Tissue Buffer and 10µl 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

DNA Extraction

1. Centrifuged all tubes at 12,000 rcf for 1 minute to pellet any debris and beads
2. Removed supernatant into new 1.5mL tubes
3. Added 1 volume (420µl) Genomic Binding Buffer to each tube, vortexed and spun down
4. 700µl of sample was added to the kit spin column and centrifuged at 12,000 rcf for 1 minute
5. Collection tubes were discarded
6. The rest of the coral samples were run through the column in the same way
7. Added 400µl DNA Pre-Wash Buffer, centrifuged at 12,000 rcf for 1 minute, and discarded the flow through
8. Added 700µl G-DNA Wash Buffer, centrifuged at 12,000 rcf for 1 minute, and discarded the flow through
9. Added 200µl G-DNA Wash Buffer, centrifuged at 12,000 rcf for 1 minute, and discarded the collection tube
10. Columns were transferred to 1.5mL tubes
11. Added 50µl warmed 70 degrees C 10mM Tris-HCl directly to the column filter and incubated at room temp for 5 minutes
12. Centrifuged for 1 minute at 12,000 rcf
13. Repeated steps 11 and 12 one more time
14. Stored tubes in fridge to qubit the next immediately

Qubit

• Broad Range dsDNA Qubit protocol
• Qubit was performed by Marygrace Trusdell
• All samples were read twice

Sample	DNA Standard 1 (RFU)	DNA Standard 2 (RFU)	DNA 1 (ng/µl)	DNA 2 (ng/µl)	Average DNA
5	232	20534	36	36.2	36.1
8	232	20534	24.6	24.2	24.4
49	232	20534	70	70.6	70.3
50	232	20534	73.4	73	73.2
67	232	20534	7.06	6.90	6.97
70	232	20534	38.8	39	38.9
192	232	20534	67.2	65.8	66.5
198	232	20534	40.8	40.8	40.8
220	232	20534	20.2	19.7	20
246	232	20534	36.2	34	35.1
338	232	20534	22.6	22	22.3
364	232	20534	46	50	48
172	232	20534	128	126	125
176	232	20534	86	86	86
186	232	20534	31.2	32.4	31.7
384	232	20534	54	54	54
405	232	20534	23.8	24.4	32.1
424	232	20534	48.6	47.8	47.2

Gel Verification

• A 1.5% agarose gel was ran to check the integrity of the genomic DNA
• Following the PPP Lab protocol
• Gel was run by Marygrace Trusdell
• Two samples are from Kevin Wong’s Astrangia homogenates

Samples 67, 220, and 338 don’t look like they are very good quality. I am going to try to re-extract from 67 and 220 because those are quite low. I should also try 338. This gel was run on the 18th, which is after I started the next extraction.