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 20, 2021

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
161	January	Pocillopora	ACR-256	20200106	3
177	January	Porites	POC-239	20200110	1
185	January	Porites	POR-354	20200110	1
417	March	Pocillopora	POC-238	20200305	1
479	March	Porites	ACR-360	20200305	1
483	March	Porites	POR-367	20200305	1
701	Sept	Acropora	POC-257	20200911	1
705	Sept	Pocillopora	POR-341	20200908	3
707	Sept	Porites	POR-79	20200908	3
919	November	Acropora	ACR-145	20201030	2
925	November	Pocillopora	POC-48	20201030	2
927	November	Porites	POR-262	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	170.40
Standard 2	23812.88
161		8.78	8.62	8.7
177		nd	nd	nd
185		nd	nd	nd
417		51.4	51.4	51.4
479		4.14	3.98	4.06
483		nd	nd	nd
701		14.7	14.4	14.55
705		59.8	59.6	59.7
707		38.6	38.0	38.3
919		27.4	27.2	27.3
925		18.8	18.3	18.55
927		19.6	19.5	19.55

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	372.42
Standard 2	6917.76
161		20.2	20.2	20.2
177		nd	nd	nd
185		20.8	20.8	20.8
417		47.6	47.2	47.4
479		nd	nd	nd
483		13.0	13.0	13.0
701		14.4	13.4	13.9
705		40.4	40.8	40.6
707		41.4	41.0	41.2
919		15.6	15.8	15.7
925		28.8	28.6	28.7
927		nd	nd	nd

Tape Station

• Used to check RNA quality Protocol
• Did not tape station 177, 479, and 927
• 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

• 927 was very mucusy in the tube