DNA/RNA extractions from E5 project

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

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Extraction Date: September 16, 2021

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
29	January	Porites	POR-216	20200103	2
33	January	Acropora	ACR-265	20200103	2
49	January	Acropora	ACR-237	20200103	2
357	March	Porites	POR-341	20200304	3
359	March	Acropora	ACR-351	20200304	3
363	March	Pocillopora	POC-366	20200304	3
589	Sept	Porites	POR-353	20200908	3
597	Sept	Pocillopora	POC-375	20200908	3
621	Sept	Acropora	ACR-364	20200908	3
735	November	Porites	POR-79	20201101	1
743	November	Acropora	ACR-176	20201101	1
747	November	Acropora	ACR-140	20201101	1

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	206.46
Standard 2	20887.01
29		3.20	3.06	3.13
33		20.6	20.0	20.3
49		18.4	18.0	18.2
357		3.78	3.62	3.7
359		17.6	17.2	17.4
363		70.8	69.0	69.9
589		10.5	10.5	10.5
597		30.8	30.0	30.4
621		50.0	50.2	50.1
735		2.64	2.52	2.58
743		43.4	43.2	43.3
747		100.0	98.4	99.2

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	371.75
Standard 2	7582.53
29		31.4	31.0	31.2
33		13.8	13.8	13.8
49		12.4	13.2	12.8
357		42.2	42.0	42.1
359		14.0	13.6	13.8
363		58.6	58.0	58.3
589		16.4	15.8	16.1
597		32.4	31.8	32.1
621		27.0	26.6	26.8
735		16.0	15.8	15.9
743		38.6	37.8	38.2
747		15.4	15.8	15.6

Tape Station

• Used to check RNA quality Protocol
• 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

• NA