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 02, 2021

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
243	January	Porites	POR-81	20200110	1
255	January	Pocillopora	POC-55	20200110	1
261	January	Acropora	ACR-51	20200110	1
269	March	Porites	POR-221	20200303	2
399	March	Pocillopora	POC-52	20200305	1
439	March	Acropora	ACR-173	20200305	1
511	Sept	Acropora	ACR-237	20200910	2
549	Sept	Porites	POR-240	20200910	2
653	Sept	Pocillopora	POC-50	20200911	1
915	November	Porites	POR-216	20201030	2
921	November	Pocillopora	POC254	20201030	2
935	November	Acropora	ACR-256	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	179.32
Standard 2	28236.78
243		nd	nd	nd
255		4.30	4.26	4.28
261		20.8	20.6	20.7
269		2.18	2.20	2.19
399		18.6	18.4	18.5
439		20.0	19.8	19.9
511		32.0	31.6	31.8
549		21.6	21.0	21.3
653		37.6	37.0	37.3
915		13.5	13.2	13.35
921		9.40	9.24	9.32
935		34.0	33.4	33.7

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	360.18
Standard 2	6437.06
243		14.8	15.2	15.0
255		nd	nd	nd
261		11.0	11.0	11.0
269		11.8	12.0	11.9
399		23.8	23.4	23.6
439		11.2	11.6	11.4
511		15.8	16.2	16.0
549		nd	10.0(?)	10.0
653		34.3	34.4	34.35
915		20.4	20.0	20.2
921		36.4	36.6	36.5
935		10.8	11.4	11.1

Tape Station

• Used to check RNA quality Protocol
• Did not tape station number 255
• 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

• 445, 593, and 877 had pigment in the final RNA elution