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 16, 2021

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
101	January	Pocillopora	POC-358	20200106	3
129	January	Acropora	ARC-390	20200106	3
151	January	Porites	POR-384	20200106	3
389	March	Pocillopora	POC-42	20200305	1
459	March	Porites	POR-385	20200304	3
473	March	Porites	POR-77	20200305	1
571	Sept	Pocillopora	POC-372	20200908	3
595	Sept	Pocillopora	POC-378	20200908	3
699	Sept	Acropora	ACR-186	20200911	1
903	November	Porites	POR-245	20201030	2
905	November	Acropora	ACR-218	20201030	2
909	November	Pocillopora	POC-200	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	191.07
Standard 2	27554.94
101		16.5	16.4	16.45
129		13.6	13.5	13.55
151		2.28	2.26	2.27
389		21.0	21.4	21.2
459		nd	nd	nd
473		3.74	3.74	3.74
571		18.9	18.9	18.9
595		2.66	2.66	2.66
699		42.2	42.4	42.3
903		4.28	4.26	4.27
905		62.0	62.2	62.1
909		3.12	3.08	3.10

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	329.23
Standard 2	6106.61
101		66.4	67.0	66.7
129		17.0	16.0	16.5
151		nd	nd	nd
389		47.8	48.4	48.1
459		13.6	14.0	13.8
473		19.8	20.0	19.9
571		57.6	57.4	57.5
595		51.6	52.4	52.0
699		18.0	16.8	17.4
903		nd	nd	nd
905		29.0	28.8	28.9
909		78.6	77.6	78.1

Tape Station

• Used to check RNA quality Protocol
• Did not tape station #151 and 903
• 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 5ul of gel red 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

• 473, and 903 had pigment in the final RNA elution
• Gel looks like absolute trash. I put blame on using gel red instead of gel green.