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 01, 2021

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
175	January	Porites	POR-70	20200110	1
213	January	Pocillopora	POC-52	20200110	1
233	January	Acropora	ACR-185	20200110	1
419	March	Pocillopora	POC-369	20200303	2
443	March	Acropora	ACR-190	20200305	1
491	March	Porites	POR-73	20200305	1
645	Sept	Porites	POR-82	20200910	2
679	Sept	Pocillopora	POC-68	20200910	2
681	Sept	Acropora	ACR-178	20200911	1
845	November	Porites	POR-384	20201030	2
887	November	Pocillopora	POC-255	20201030	2
897	November	Acropora	ACR-210	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	22334.53
175		nd	nd	nd
213		5.40	5.26	5.33
233		27.2	26.8	27.0
419		59.8	59.6	59.7
443		30.4	29.4	29.9
491		2.16	2.06	2.11
645		3.46	3.46	3.46
679		40.4	39.8	40.1
681		56.2	55.2	55.7
845		57.8	56.8	57.3
887		10.9	10.5	10.7
897		41.0	41.2	41.1

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	369.37
Standard 2	6757.74
175		nd	nd	nd
213		nd	nd	nd
233		14.2	14.6	14.4
419		64.6	64.2	64.4
443		10.2	10.4	10.3
491		26.6	26.4	26.5
645		34.6	33.4	34.0
679		71.0	73.4	72.2
681		18.6	18.6	18.6
845		14.6	15.2	14.9
887		48.8	48.2	48.5
897		17.2	16.8	17.0

Tape Station

• Used to check RNA quality Protocol
• Did not tape station #175
• 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