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 8, 2021

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
93	January	Acropora	ACR-364	20200106	3
95	January	Pocillopora	POC-372	20200106	3
125	January	Porites	POR-355	20200106	3
283	March	Porites	POR-209	20200303	2
323	March	Pocillopora	POC-259	20200303	2
373	March	Pocillopora	POC-359	20200304	3
581	Sept	Pocillopora	POC-359	20200908	3
599	Sept	Porites	POR-367	20200908	3
609	Sept	Porites	POR-384	20200908	3
875	November	Pocillopora	POC-207	20201030	2
895	November	Acropora	ACR-234	20201030	2
907	November	Porites	POR-221	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	203.34
Standard 2	22004.74
93		8.52	8.48	8.50
95		33.8	33.6	33.7
125		nd	nd	nd
283		4.44	4.36	4.40
323		36.6	36.4	36.5
373		42.4	41.8	42.1
581		70.2	70.6	70.3
599		2.32	2.32	2.32
609		36.0	36.2	36.1
875		14.0	13.8	13.9
895		83.6	82.8	83.2
907		8.26	8.30	8.28

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	367.98
Standard 2	6519.33
93		nd	nd	nd
95		nd	nd	nd
125		10.2	10.0	10.1
283		nd	nd	nd
323		35.6	35.6	35.6
373		63.8	63.2	63.5
581		39.0	37.4	38.2
599		20.4	20.2	20.3
609		12.8	12.6	12.7
875		40.6	40.4	40.5
895		18.8	18.4	18.6
907		nd	nd	nd

Tape Station

• Used to check RNA quality Protocol
• Diod not tape station #93, 95, 283, and 907
• 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

• 125 and 599 had pigment in the final RNA elution