DNA/RNA extractions from E5 project

Extractions from the three coral species from each of the four timepoints

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Extraction Date: November 4, 2021

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
17	January	Porites	POR-221	20200103	2
79	January	Pocillopora	POC-207	20200103	2
85	January	Acropora	ACR-351	20200106	3
287	March	Porites	POR-235	20200303	2
317	March	Pocillopora	POC-239	20200303	2
435	March	Acropora	ACR-51	20200303	1
515	Sept	Porites	POR-216	20200910	2
573	Sept	Pocillopora	POC-358	20200908	3
685	Sept	Acropora	ACR-190	20200911	1
883	November	Porites	POR-224	20201030	2
899	November	Pocillopora	POC-205	20201030	2
911	November	Acropora	ACR-225	20201030	2

Extraction notes

• ACR and POC samples: pulled out 300ul of shield
• POR samples: pulled out 150ul of shield and added to 150ul of new shield
• Spun down samples for 3 minutes at 9000 rcf and then transfer the supernatant to new tube without disturbing the pellet
• 300ul of shield, 15ul of ProK, and 30ul of ProK digestion buffer, let sit for 2 minutes
• All spins were done for 1 minute or 2.30 minutes
• Did two washes with 700ul of wash buffer for both the DNA and RNA
• Then followed the protocol as described in protocol

Qubit

• Used Broad range dsDNA and RNA Qubit Protocol
• All samples read twice, standard only read once

DNA

Tube number	RFU	DNA 1 (ng/uL)	DNA 2 (ng/uL)	Average
Standard 1	208.38
Standard 2	32378.83
17		2.12	2.08	2.10
79		20.2	20.6	20.4
85		13.6	13.5	13.55
287		nd	nd	nd
317		90.6	89.4	90.0
435		17.7	17.6	17.65
515		2.32	2.30	2.31
573		44.2	43.6	43.9
685		48.4	47.8	48.1
883		21.6	21.6	21.6
899		12.1	12.0	12.05
911		56.6	55.6	56.1

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	350.59
Standard 2	7152.30
17		11.2	11.2	11.2
79		22.4	22.4	22.4
85		nd	nd	nd
287		15.6	15.2	15.4
317		43.2	43.4	43.3
435		13.8	14.6	14.2
515		nd	nd	nd
573		44.6	44.4	44.5
685		20.4	20.2	20.3
883		19.4	19.0	19.2
899		77.2	77.0	77.1
911		17.6	17.4	17.5

Tape Station

• Used to check RNA quality Protocol
• Did not tape station #85 and #515
• Results Link

Gel

• Modified from this protocol
• Added 0.75g of agarose and 50ml of 1x TAE to flask and microwaved for 45 seconds. This makes a 1.5% gel
• Once cool enough to touch added 2ul of gel green stain
• Swirled and poured into gel mould with comb
• Once solidified, covered with 1X TAE as a running buffer
• Added 1ul of purple loading dye to each of my QC strip tube samples. I had ~9ul of DNA leftover from QC and ~8ul of RNA
• Loaded my gel with the DNA first, then skipped a well and then the RNA
• Ran the gel for 60 minutes at 60 volts

Addtional Notes

• 287, and 515 had pigment in the final RNA elution
• something went a little funky when loading the RNA gel. Take with a grain of salt. Maybe will rerun.