DNA/RNA extractions from E5 project

Extractions from the three coral species from the first two timepoints

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Extraction Date: November 29, 2021

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
173	January	Porites	POC-346	20200106	3
183	January	Pocillopora	ACR-396	20200106	3
189	January	Pocillopora	POR-383	20200106	3
193	January	Porites	POC-257	20200303	2
197	January	Acropora	POR-253	20200303	2
239	January	Pocillopora	POR-266	20200303	2
251	January	Porites	POC-371	20200908	3
257	January	Pocillopora	POR-362	20200908	3
259	January	Pocillopora	ACR-210	20200910	2
403	March	Pocillopora	POC-55	20201101	1
449	March	Pocillopora	POR-240	20201030	2
489	March	Porites	ACR-229	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	190.38
Standard 2	29110.40
173		nd	nd	nd
183		12.2	12.1	12.15
189		10.6	10.7	10.65
193		nd	nd	nd
197		7.26	7.08	7.17
239		9.82	9.66	9.74
251		nd	nd	nd
257		13.5	13.3	13.4
259		12.5	12.2	12.35
403		29.0	29.2	29.1
449		21.6	21.4	21.5
489		3.76	3.68	3.72

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	411.31
Standard 2	13430.28
173		10.8	10.8	10.8
183		12.4	12.6	12.5
189		17.4	17.6	17.5
193		16.4	16.2	16.3
197		nd	nd	nd
239		nd	nd	nd
251		nd	nd	nd
257		13.2	13.4	13.3
259		10.8	10.8	10.8
403		27.6	27.4	27.5
449		24.6	24.4	24.5
489		28.2	28.6	28.4

Tape Station

• Used to check RNA quality Protocol
• Did not tape station #251
• 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

• 489 had pigment in the final RNA elution
• 259 was written on the cap, but 260 on the tube
• Accidentally eluted 189 in 150ul of Tris