DNA/RNA extractions from E5 project

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

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Extraction Date: September 13, 2021

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
165	January	Porites	POR-362	20200106	3
171	January	Porites	POR-70	20200106	3
181	January	Porites	POR-77	20200110	1
333	March	Porites	POR-242	20200303	2
361	March	Porites	POR-355	20200304	3
371	March	Porites	POR-338	20200304	3
557	Sept	Porites	POR-260	20200910	2
579	Sept	Porites	POR-381	20200908	3
585	Sept	Porites	POR-354	20200908	3
749	November	Porites	POR-74	20201101	1
787	November	Porites	POR-338	20201031	3
823	November	Porites	POR-365	20201031	3

Extraction notes

• POR samples: pulled out 150ul of shield from the original sample tube, and added that to a new 1.5ml tube containing 150ul of new RNA/DNA 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	194.99
Standard 2	21637.60
165		nd	nd	nd
171		nd	nd	nd
181		nd	nd	nd
333		2.38	2.24	2.31
361		nd	nd	nd
371		nd	nd	nd
557		nd	nd	nd
579		nd	nd	nd
585		nd	nd	nd
749		nd	nd	nd
787		nd	nd	nd
823		2.98	2.26	2.62

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	423.39
Standard 2	10648.06
165		15.0	14.6	14.8
171		22.6	22.2	22.4
181		15.6	14.8	15.2
333		21.4	21.0	21.2
361		22.4	22.2	22.3
371		21.2	21.4	21.3
557		18.2	18.4	18.3
579		nd	nd	nd
585		18.8	18.2	18.5
749		33.8	33.4	33.6
787		13.4	13.2	13.3
823		16.6	16.8	16.7

Tape Station

• Samples not analyzed

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

• Nearly all RNA elute had some pigment. The RNA spin columns were also pigmented.