DNA/RNA extractions from E5 project

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

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Extraction Date: September 7, 2021

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
19	January	Porites	POR-224	20200103	2
43	January	Acropora	ACR-246	20200103	2
61	January	Pocillopora	POC-254	20200103	2
313	March	Porites	POR-214	20200304	2
331	March	Acropora	ACR-265	20200304	2
345	March	Pocillopora	POC-371	20200305	3
551	Sept	Porites	POR-242	20200910	2
617	Sept	Acropora	ACR-343	20200908	3
619	Sept	Pocillopora	POC-373	20200908	3
751	November	Porites	POR-73	20201101	1
759	November	Acropora	ACR-185	20201101	1
773	November	Pocillopora	POC-44	20201101	1

Extraction notes

• ACR and POC samples: pulled out 300ul of shield
• POR samples: pulled out 150ul and then added 150ul of new shield. The thought behind this was to try and limit the amount of inhibitor
• 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	195.31
Standard 2	20691.61
19		2.44	2.4	2.42
43		18.9	18.9	18.9
61		10.3	10.3	10.3
313		5.48	5.5	5.49
331		42.2	42.2	42.2
345		21.6	21.8	21.7
551		6.68	6.62	6.65
617		66.2	65.4	65.8
619		28.4	28.4	28.4
751		4.74	4.76	4.75
759		18.1	17.8	17.95
773		60.2	60.0	60.1

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	348.62
Standard 2	7004.32
19		nd	nd	nd
43		11.4	10.0	10.7
61		29.4	29.2	29.3
313		12.4	11.8	12.1
331		30.4	29.8	30.1
345		74.4	73.4	73.9
551		15.2	15.0	15.1
617		26.0	26.4	26.2
619		37.0	37.0	37.0
751		14.4	14.6	14.5
759		26.2	26.2	26.2
773		61.0	61.0	61.0

Tape Station

• Used to check RNA quality Protocol
• I did not tape station 19 because there was no detectable RNA
• 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

• RNA gel doesn’t look great. Tape station looks a little better