DNA/RNA extractions from E5 project

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

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Extraction Date: September 30, 2021

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
135	January	Porites	POR-242	20200103	2
141	January	Acropora	ACR-234	20200103	2
149	January	Pocillopora	POC-222	20200103	2
289	March	Porites	POR-260	20200303	2
295	March	Pocillopora	POC-201	20200303	2
297	March	Acropora	ACR-347	20200304	3
559	Sept	Porites	POR-72	20200911	1
623	Sept	Pocillopora	POC-48	20200911	1
625	Sept	Pocillopora	POC-43	20200911	1
829	November	Porites	POR-353	20201031	3
833	November	Pocillopora	POC-358	20201031	3
837	November	Acropora	ACR-393	20201031	3

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	213.55
Standard 2	21639.23
11		5.48	5.50	5.49
35		51.2	51.0	51.1
65		44.0	42.6	43.3
327		2.08	2.06	2.07
329		84.0	83.8	83.9
337		9.46	9.39	9.43
637		nd	nd	nd
643		64.2	65.4	64.8
655		60.8	61.2	61.0
849		13.7	13.5	13.6
851		4.52	4.54	4.53
855		9.98	9.78	9.88

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	368.89
Standard 2	6287.72
11		17.0	16.8	16.9
35		18.6	18.4	18.5
65		35.4	37.0	36.2
327		28.2	29.0	28.6
329		57.2	59.4	58.3
337		14.6	14.6	14.6
637		29.0	29.4	29.2
643		53.6	53.0	53.3
655		42.4	40.8	41.6
849		13.8	14.2	14.0
851		59.4	58.8	59.1
855		17.8	17.6	17.7

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

• 327 some pigment carryover
• Even though qubit detected no gDNA in 637, there’s a band in the gel