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 15, 2021

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
51	January	Acropora	ACR-258	20200103	2
81	January	Pocillopora	POC-217	20200103	2
115	January	Porites	POR-385	20200106	3
407	March	Pocillopora	POC-41	20200305	1
471	March	Porites	POR-76	20200305	1
477	March	Porites	POR-69	20200305	1
587	Sept	Pocillopora	POC-366	20200908	3
613	Sept	Porites	POR-365	20200908	3
669	Sept	Porites	POR-83	20200911	1
779	November	Pocillopora	POC-57	20201101	1
861	November	Acropora	ACR-213	20201030	2
867	November	Porites	POR-209	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	204.54
Standard 2	22583.17
51		28.8	28.4	28.6
81		30.6	30.4	30.5
115		2.46	2.42	2.44
407		43.0	43.0	43.0
471		2.76	2.64	2.70
477		2.22	2.14	2.18
587		48.4	48.2	48.3
613		3.04	2.96	3.0
669		2.12	nd	2.12
779		5.86	5.94	5.9
861		97.4	95.8	96.6
867		6.54	6.40	6.47

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	375.55
Standard 2	6920.82
51		15.2	15.4	15.3
81		nd	nd	nd
115		11.4	10.8	11.1
407		45.2	45.2	45.2
471		20.0	20.4	20.2
477		13.2	13.4	13.3
587		32.4	32.4	32.4
613		19.6	20.2	19.9
669		12.4	12.0	12.2
779		29.8	30.6	30.2
861		13.8	13.8	13.8
867		nd	nd	nd

Tape Station

• Used to check RNA quality Protocol
• Did not tape station #81
• 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

• There is a band in 81 RNA even though qubit said none
• 471, and 669 had pigment in the final RNA elution