DNA/RNA extractions from E5 project

Extractions from the three coral species from timepoints 1, 2 and 4

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Extraction Date: November 22, 2021

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
133	January	Pocillopora	POC-395	20200106	3
137	January	Pocillopora	POC-366	20200106	3
157	January	Porites	POR-353	20200106	3
447	March	Pocillopora	POC-395	20200304	3
469	March	Porites	POR-83	20200305	3
487	March	Porites	POR-71	20200305	1
879	November	Pocillopora	POC-222	20201030	1
885	November	Acropora	ACR-241	20201030	2
913	November	Pocillopora	POC-248	20201030	2
917	November	Acropora	ACR-220	20201030	2
929	November	Acropora	ACR-237	20201030	2
933	November	Pocillopora	POC-239	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	193.96
Standard 2	21317.41
133		31.0	30.8	30.9
137		39.0	38.2	38.6
157		5.92	5.84	5.88
447		18.4	18.2	18.3
469		nd	nd	nd
487		2.34	2.28	2.31
879		7.16	7.0	7.08
885		38.0	37.2	37.6
913		6.36	6.16	6.26
917		45.0	44.8	44.9
929		115	114	114.5
933		11.2	11.0	11.1

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	420.08
Standard 2	10880.55
133		18.8	18.4	18.6
137		17.0	17.2	17.1
157		nd	nd	nd
447		21.0	20.6	20.8
469		17.6	17.8	17.7
487		26.4	27.0	26.7
879		14.2	14.2	14.2
885		nd	nd	nd
913		23.8	23.6	23.7
917		21.6	21.4	21.5
929		13.0	12.6	12.8
933		22.6	22.4	22.5

Tape Station

• Used to check RNA quality Protocol
• Did not tape station #157
• 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

• 469 and 487 had pigment in the final RNA elution