DNA/RNA extractions from E5 project

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

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Extraction Date: March 1, 2022

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
165	January	Porites	POR-362	20200106	3
171	January	Porites	POR-70	20200106	3
181	January	Porites	POR-77	20200110	1
361	March	Porites	POR-355	20200304	3
371	March	Porites	POR-338	20200304	3
557	Sept	Porites	POR-260	20200910	2
579	Sept	Porites	POR-381	20200908	3
585	Sept	Porites	POR-354	20200908	3
749	November	Porites	POR-74	20201101	1
787	November	Porites	POR-338	20201031	3
923	November	Porites	POR-240	20201031	3
927	November	Porites	POR-253	20201031	3

Extraction notes

• I looked at each sample, for those samples that were darker (209, 353, 475, and 843) I took 100ul of sample and added it to 200ul of new shield
• For samples that were lighter in pigment, (19, 97, 149, 191, 543, 591, and 867) I took 200ul of sample and added to 100ul 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 10 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	192.47
Standard 2	20809.40
165		6.58	6.40	6.49
171		4.36	4.34	4.35
181		2.30	2.28	2.29
333		2.68	2.64	2.66
361		2.68	2.54	2.61
371		2.06	2.02	2.04
557		5.68	5.58	5.63
579		2.10	2.00	2.05
585		2.80	2.76	2.78
749		2.62	2.60	2.61
787		38.2	37.6	37.9
823		24.4	24.2	24.3

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	420.48
Standard 2	10534.41
165		30.8	30.6	30.7
171		26.2	26.8	26.5
181		20.2	19.8	20.0
333		22.6	22.6	22.6
361		29.6	30.0	29.8
371		15.6	15.8	15.7
557		nd	nd	nd
579		15.6	15.6	15.6
585		27.2	26.8	27.0
749		25.8	26.2	26.0
787		16.4	16.6	16.5
823		14.2	14.4	14.3

Tape Station

• Samples not analyzed

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

• 165, 171, 361, 749, and 787 all had pigmentation in the final RNA elution
• 923 and 927 and badly degraded