DNA/RNA extractions from E5 project

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

---

Extraction Date: February 24, 2022

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
131	January	Porites	POR-357	20200106	3
151	January	Porites	POR-384	20200106	3
157	January	Porites	POR-353	20200110	1
163	January	Porites	POR-340	20200304	3
167	January	Porites	POR-383	20200304	3
173	January	Porites	POR-349	20200910	2
175	January	Porites	POR-70	20200908	3
177	January	Porites	POR-78	20200908	3
185	January	Porites	POR-82	20201101	1
193	January	Porites	POR-75	20201031	3
195	January	Porites	POR-69	20201031	3
807	November	Porites	POR-387	20201031	3

Extraction notes

• I looked at each sample, for those samples that were darker (167, and 807) I took 100ul of sample and added it to 200ul of new shield
• For samples that were lighter in pigment, (131, 151, 157, 163, 173, 175, 177, 185, 193, and 195) I took 200ul of sample and added to 100ul 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	191.07
Standard 2	20741.72
131		2.58	2.44	2.51
151		4.78	4.64	4.71
157		7.68	7.60	7.64
163		2.32	2.26	2.29
167		nd	nd	nd
173		2.94	2.88	2.91
175		nd	nd	nd
177		nd	nd	nd
185		3.12	3.12	3.12
193		2.64	2.66	2.65
195		2.14	2.10	2.12
807		2.14	2.10	2.12

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	242.15
Standard 2	10674.37
131		nd	nd	nd
151		nd	nd	nd
157		11.4	10.6	11.0
163		20.0	19.8	19.9
167		13.8	13.8	13.8
173		18.2	17.8	18.0
175		nd	nd	nd
177		nd	nd	nd
185		31.0	31.0	31.0
193		22.0	23.2	22.6
195		19.4	19.2	19.3
807		17.4	17.2	17.3

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

• 185 and 193 had some pigmentation in the final RNA elution