DNA/RNA extractions from E5 project

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

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Extraction Date: October 12, 2021

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
15	January	Porites	POR-251	20200103	2
37	January	Acropora	ACR-243	20200103	2
71	January	Pocillopora	POC-248	20200103	2
379	March	Porites	POR-383	20200304	3
385	March	Pocillopora	POC-47	20200304	1
427	March	Acropora	ACR-145	20200305	1
591	Sept	Porites	POR-555	20200908	3
659	Sept	Pocillopora	POC-52	20200911	1
675	Sept	Acropora	ACR-140	20200911	1
723	November	Porites	POR-82	20201101	1
729	November	Pocillopora	POC-57	20201101	1
817	November	Acropora	ACR-398	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	195.82
Standard 2	22265.51
15		nd	nd	nd
37		62.2	62.6	62.4
71		48.6	48.6	48.6
379		2.46	2.46	2.46
385		25.6	25.4	25.5
427		19.6	19.6	19.6
591		nd	nd	nd
659		60.2	60.4	60.3
675		45.6	45.6	45.6
723		2.82	2.76	2.79
729		50.8	51.0	50.9
817		19.0	18.8	18.9

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	379.41
Standard 2	7092.84
15		nd	nd	nd
37		13.2	14.2	13.7
71		38.4	39.2	38.8
379		35.0	35.2	35.1
385		31.8	32.0	31.9
427		20.8	20.8	20.8
591		12.6	13.4	13.0
659		45.2	45.6	45.4
675		14.0	14.4	14.2
723		32.4	32.6	32.5
729		32.2	32.8	32.5
817		nd	nd	nd

Tape Station

• Used to check RNA quality Protocol
• Did not tape station #15
• 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

• 379 and 723 had pigment in the final RNA elution