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 18, 2021

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
45	January	Acropora	ACR-256	20200103	2
69	January	Pocillopora	POC-239	20200103	2
147	January	Porites	POR-354	20200106	3
321	March	Pocillopora	POC-238	20200303	2
343	March	Acropora	ACR-360	20200304	3
445	March	Porites	POR-367	20200304	3
561	Sept	Pocillopora	POC-257	20200910	2
593	Sept	Porites	POR-341	20200908	3
635	Sept	Porites	POR-79	20200911	1
725	November	Acropora	ACR-145	20201101	1
727	November	Pocillopora	POC-48	20201101	1
877	November	Porites	POR-262	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	197.12
Standard 2	22201.70
45		20.2	20.2	20.2
69		28.2	28.2	28.2
147		2.74	2.76	2.75
321		162	162	162
343		4.70	4.64	4.67
445		2.38	2.24	2.31
561		15.7	15.6	15.65
593		2.90	2.82	2.86
635		9.06	8.96	9.02
725		66.6	66.6	66.6
727		85.0	84.2	84.3
877		3.76	3.64	3.70

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	360.18
Standard 2	6437.06
45		14.4	14.8	14.6
69		27.0	27.4	27.2
147		19.0	18.2	18.6
321		31.8	31.8	31.8
343		nd	nd	nd
445		25.2	24.6	24.9
561		47.0	47.2	47.1
593		29.4	30.8	30.1
635		19.4	19.2	19.3
725		19.4	18.8	19.1
727		45.6	44.6	45.1
877		36.6	36.4	36.5

Tape Station

• Used to check RNA quality Protocol
• 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