DNA/RNA extractions from E5 project

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

---

Extraction Date: November 5, 2021

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
7	January	Porites	POR-262	20200103	2
13	January	Porites	POR-245	20200103	2
75	January	Pocillopora	POC-201	20200103	2
293	March	Porites	POR-224	20200303	2
309	March	Pocillopora	POC-207	20200303	2
387	March	Pocillopora	POC-44	20200305	1
497	Sept	Porites	PO4-253	20200910	2
531	Sept	Acropora	ACR-229	20200910	2
563	Sept	Pocillopora	POC-254	20200910	2
763	November	Acropora	ACR-150	20201101	1
795	November	Porites	POR-354	20201031	3
811	November	Pocillopora	POC-360	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	201.64
Standard 2	22210.39
7		nd	nd	nd
13		nd	nd	nd
75		38.6	38.2	38.4
293		3.80	3.16	3.48
309		63.0	61.8	62.4
387		49.2	48.6	48.9
497		10.6	10.5	10.55
531		72.2	71.6	71.9
563		27.4	27.4	27.4
763		56.2	55.0	55.6
795		nd	nd	nd
811		72.8	69.6	71.2

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	360.18
Standard 2	6437.06
7		13.0	13.0	13.0
13		11.0	11.0	11.0
75		23.2	23.4	23.3
293		12.6	12.8	12.7
309		42.0	42.2	42.1
387		35.2	34.6	34.9
497		nd	nd	nd
531		17.4	17.6	17.5
563		20.4	20.2	20.3
763		17.6	17.0	17.3
795		12.8	12.4	12.6
811		48.0	49.0	48.5

Tape Station

• Used to check RNA quality Protocol
• Did not tape station #497
• 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

• 795 had pigment in the final RNA elution