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 01, 2021

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
21	January	Porites	POR-235	20200103	2
55	January	Acropora	ACR-229	20200103	2
139	January	Acropora	ACR-393	20200106	3
381	March	Porites	POR-365	20200304	3
383	March	Pocillopora	POC-45	20200305	1
405	March	Pocillopora	POC-68	20200305	1
641	Sept	Porites	POR-74	20200911	1
657	Sept	Pocillopora	POC-57	20200911	1
695	Sept	Pocillopora	POC-47	20200911	1
745	November	Porites	POR-75	20201101	1
761	November	Acropora	ACR-139	20201101	1
799	November	Pocillopora	POC-372	20201031	3

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	193.55
Standard 2	21842.45
21		nd	nd	nd
55		31.0	29.8	30.4
139		19.3	19.1	19.2
381		5.36	5.36	5.36
383		48.8	48.2	48.5
405		23.6	23.6	23.6
641		2.82	2.74	2.78
657		49.0	49.0	49.0
695		56.4	56.0	56.2
745		5.80	5.66	5.73
761		47.4	47.0	47.2
799		64.0	63.2	63.6

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	378.09
Standard 2	7098.82
21		13.2	13.2	13.2
55		10.2	11.6	10.9
139		13.6	13.0	13.3
381		31.4	32.3	31.85
383		41.6	42.2	41.9
405		28.2	27.8	28.0
641		14.6	14.6	14.6
657		51.2	50.8	51.0
695		49.0	49.2	49.1
745		nd	nd	nd
761		14.0	13.6	13.8
799		42.6	42.4	42.5

Tape Station

• Used to check RNA quality Protocol
• Did not tape station 745
• 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

• 381, 641, 745 some pigment carryover
• Even though qubit did not detect any gDNA in 21, there is a band in the gel
• Qubit also did not detect any RNA in 745, but there is a good band in the gel