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 28, 2021

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
27	January	Porites	POR-214	20200103	2
83	January	Pocillopora	POC-219	20200106	2
91	January	Acropora	ACR-363	20200106	3
395	March	Pocillopora	POC-56	20200305	1
431	March	Acropora	ACR-139	20200305	1
453	March	Porites	POR-354	20200304	3
505	Sept	Acropora	ACR-265	20200910	2
545	Sept	Pocillopora	POC-259	20200910	2
629	Sept	Porites	POR-349	20200908	3
731	November	Acropora	ACR-180	20201101	1
733	November	Pocillopora	POC-47	20201101	1
739	November	Porites	POR-70	20201101	1

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	199.37
Standard 2	22993.16
27		6.08	6.18	6.13
83		13.7	13.8	13.75
91		21.0	20.8	20.9
395		32.4	32.6	32.5
431		36.6	36.6	36.6
453		2.60	2.60	2.60
505		19.8	19.4	19.6
545		9.58	9.48	9.53
629		nd	nd	nd
731		44.0	43.2	43.6
733		33.0	33.0	33.0
739		nd	nd	nd

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	374.67
Standard 2	7085.21
27		18.6	18.8	18.7
83		32.2	32.6	32.4
91		12.6	12.6	12.6
395		37.6	37.4	37.5
431		13.4	13.0	13.2
453		40.4	40.2	40.3
505		10.4	10.4	10.4
545		22.2	22.2	22.2
629		15.8	15.8	15.8
731		14.6	14.0	14.3
733		20.0	19.8	19.9
739		17.2	17.0	17.1

Tape Station

• Used to check RNA quality Protocol
• 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 are bands in the DNA gel for 629 and 739