DNA/RNA extractions from E5 project

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

---

Extraction Date: November 15, 2021

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
179	January	Pocillopora	POC-68	20200110	1
187	January	Acropora	ACR-145	20200110	1
191	January	Porites	POR-71	20200110	1
279	March	Porites	POR-206	20200303	2
335	March	Pocillopora	POC-215	20200303	2
377	March	Pocillopora	POC-346	20200304	3
575	Sept	Pocillopora	POC-395	20200908	3
647	Sept	Porites	POR-71	20200911	1
683	Sept	Pocillopora	POC-45	20200911	1
827	November	Pocillopora	POC-395	20201031	3
863	November	Acropora	ACR-267	20201030	2
901	November	Porites	POR-253	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	314.79
Standard 2	23313.39
179		59.4	58.8	59.1
187		18.1	17.8	17.95
191		nd	nd	nd
279		2.62	2.50	2.56
335		103	102	102.5
377		28.6	28.6	28.6
575		47.4	47.6	47.5
647		nd	nd	nd
683		24.8	24.8	24.8
827		9.32	9.30	9.31
863		115	114	114.5
901		32.4	32.0	32.2

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	356.98
Standard 2	6251.85
179		44.4	47.0	45.7
187		nd	nd	nd
191		11.8	12.8	12.3
279		12.4	12.8	12.6
335		47.2	47.6	47.4
377		24.8	24.8	24.8
575		43.6	43.0	43.3
647		20.2	21.4	20.8
683		39.8	39.8	39.8
827		63.8	63.0	63.4
863		24.8	24.2	24.5
901		nd	nd	nd

Tape Station

• Used to check RNA quality Protocol
• Did not tape station #901
• 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

• 647 had pigment in the final RNA elution