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 19, 2021

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
219	January	Acropora	ACR-165	20200110	3
221	January	Pocillopora	POC-43	20200110	1
223	January	Porites	POR-80	20200110	1
311	March	Pocillopora	POC-254	20200303	1
351	March	Porites	POR-357	20200304	1
365	March	Acropora	ACR-393	20200304	1
565	Sept	Pocillopora	POC-239	20200910	1
603	Sept	Porites	POR-341	20200908	3
663	Sept	Porites	POR-70	20200911	3
857	November	Pocillopora	POC-257	20201030	2
871	November	Acropora	ACR-228	20201030	2
889	November	Porites	POR-260	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	204.60
Standard 2	22614.72
219		7.54	7.40	7.47
221		24.2	24.4	24.3
223		3.36	3.40	3.38
311		94.6	93.6	94.1
351		2.08	nd	2.08
365		16.6	16.5	16.55
565		4.24	4.16	14.2
603		2.68	2.68	2.68
663		2.92	2.82	2.87
857		41.0	41.0	41.0
871		82.2	80.6	81.4
889		6.28	6.14	6.21

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	368.75
Standard 2	7092.70
219		nd	nd	nd
221		19.0	19.0	19.0
223		15.4	15.8	15.6
311		52.8	53.8	53.3
351		23.8	23.8	23.8
365		12.0	11.8	11.9
565		26.2	26.4	26.3
603		22.6	22.6	22.6
663		10.8	10.2	10.5
857		35.8	36.0	35.9
871		18.6	18.4	18.5
889		25.8	25.8	25.8

Tape Station

• Used to check RNA quality Protocol
• Did not tape station #219
• 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

• 351, 603, 663, and 889 had pigment in the final RNA elution