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 14, 2021

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
9	January	Porites	POR-240	20200103	2
47	January	Acropora	ACR-228	20200103	2
89	January	Pocillopora	POC-359	20200106	3
425	March	Porites	POR-80	20200305	1
429	March	Acropora	ACR-286	20200305	1
437	March	Pocillopora	POC-358	20200304	3
529	Sept	Porites	POR-262	20200910	2
635	Sept	Porites	POR-79	20200911	1
687	Sept	Acropora	ACR-145	20200911	1
843	November	Porites	POR-381	20201031	3
859	November	Porites	POR-214	20201030	2
865	November	Acropora	ACR-244	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	183.89
Standard 2	21453.75
9		nd	nd	nd
47		14.6	14.1	14.35
89		31.8	32.0	31.9
425		3.94	3.82	3.88
429		42.6	42.0	42.3
437		11.9	11.7	11.8
529		2.44	2.34	2.39
635		7.22	7.00	7.11
687		47.2	46.0	46.6
843		21.2	20.4	20.8
859		11.5	11.2	11.35
865		112	111	111.5

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	367.76
Standard 2	6576.48
9		nd	nd	nd
47		10.4	nd(?)	10.4
89		30.0	29.8	29.9
425		35.0	34.0	34.5
429		13.6	14.6	14.1
437		33.8	34.2	34.0
529		19.6	19.2	19.4
635		nd	nd	nd
687		16.6	16.2	16.4
843		nd	nd	nd
859		19.4	18.8	19.1
865		18.2	18.2	18.2

Tape Station

• Used to check RNA quality Protocol
• Did not tape station #9,and 843
• 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

• 425, 529, and 635 had pigment in the final RNA elution