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 09, 2021

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
87	January	Pocillopora	POC-394	20200106	3
107	January	Acropora	ARC-345	20200106	3
113	January	Acropora	ACR-350	20200106	3
367	March	Pocillopora	POC-391	20200304	3
485	March	Porites	POR-70	20200305	1
493	March	Porites	POR-72	20200305	1
633	Sept	Porites	POR-73	20200911	1
665	Sept	Pocillopora	POC-44	20200911	1
697	Sept	Porites	POR-76	20200911	1
785	November	Pocillopora	POC-375	20201031	3
841	November	Pocillopora	POC-386	20201031	3
893	November	Porites	POR-242	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	183.85
Standard 2	21231.49
87		30.6	30.4	30.5
107		20.6	21.0	20.8
113		10.0	9.82	9.91
367		21.0	20.6	20.8
485		2.02	nd	2.00
493		2.22	2.22	2.22
633		4.52	4.46	4.49
665		55.2	54.0	54.6
697		3.30	3.16	3.23
785		60.6	60.8	60.7
841		7.60	7.44	7.52
893		20.6	20.4	20.5

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	329.23
Standard 2	6106.61
87		40.6	41.2	40.9
107		12.0	12.4	12.2
113		10.0	11.4	10.7
367		25.6	26.8	26.2
485		15.8	14.8	15.3
493		22.4	23.0	22.7
633		nd	nd	nd
665		50.8	50.6	50.7
697		21.4	21.6	21.5
785		48.4	48.6	48.5
841		66.6	65.2	65.9
893		14.8	14.8	14.8

Tape Station

• Used to check RNA quality Protocol
• Did not tape station #633
• 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

• 485, 633, and 697 had pigment in the final RNA elution
• Gel looks like absolute trash. I put blame on using gel red instead of gel green.