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 20, 2021

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
53	January	Acropora	ACR-231	20200103	2
67	January	Pocillopora	POC-238	20200103	2
111	January	Porites	POR-381	20200106	3
341	March	Acropora	ACR-368	20200304	3
355	March	Pocillopora	POC-373	20200304	3
375	March	Porites	POR-349	20200304	3
527	Sept	Acropora	ACR-225	20200910	2
535	Sept	Pocillopora	POC-201	20200910	2
541	Sept	Porites	POR-209	20200910	2
767	November	Acropora	ACR-190	20201101	1
777	November	Pocillopora	POC-50	20201101	1
797	November	Porites	POR-340	20201031	3

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	184.72
Standard 2	18696.43
53		17.8	18.1	17.95
67		44.6	45.2	44.9
111		2.28	2.42	2.35
341		15.6	15.8	15.7
355		4.64	4.94	4.79
375		22.4	22.6	22.5
527		53.0	52.8	52.9
535		17.8	18.3	18.05
541		16.2	16.1	16.15
767		68.6	67.0	68.8
777		33.2	33.2	33.2
797		2.46	2.46	2.46

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	364.46
Standard 2	7066.31
53		10.6	10.6	10.6
67		33.2	33.0	33.1
111		nd	nd	nd
341		11.0	11.2	11.1
355		38.6	38.6	38.6
375		41.6	41.4	41.5
527		19.6	20.0	19.8
535		84.6	84.0	84.3
541		11.6	11.6	11.6
767		17.8	18.6	18.2
777		57.4	56.6	57.0
797		22.0	21.2	21.6

Tape Station

• Used to check RNA quality Protocol
• Did not tape station #111
• 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

• 375 and 797 had some pigment carryover