DNA/RNA extractions from E5 project

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

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Extraction Date: November 12, 2021

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
99	January	Pocillopora	POC-346	20200106	3
121	January	Acropora	ACR-396	20200106	3
167	January	Porites	POR-383	20200106	3
267	March	Pocillopora	POC-257	20200303	2
271	March	Porites	POR-253	20200303	2
285	March	Porites	POR-266	20200303	2
569	Sept	Pocillopora	POC-371	20200908	3
601	Sept	Porites	POR-362	20200908	3
703	Sept	Acropora	ACR-210	20200910	2
753	November	Pocillopora	POC-55	20201101	1
923	November	Porites	POR-240	20201030	2
931	November	Acropora	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	190.79
Standard 2	30106.55
99		12.6	12.5	12.55
121		8.58	8.42	8.50
167		nd	nd	nd
267		nd	nd	nd
271		3.48	3.42	3.46
285		3.36	3.38	3.37
569		20.6	20.8	20.7
601		nd	nd	nd
703		15.8	15.8	15.8
753		91.8	90.4	91.1
923		20.6	20.2	20.4
931		75.6	74.6	75.1

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	370.05
Standard 2	7209.02
99		17.6	18.0	17.8
121		10.4	10.6	10.5
167		19.8	19.8	19.8
267		27.6	26.8	27.2
271		nd	nd	nd
285		11.4	11.2	11.3
569		31.0	31.4	31.2
601		10.0	nd	10.0
703		14.0	14.8	14.4
753		53.4	53.2	53.3
923		10.2	nd	nd
931		11.0	11.2	11.1

Tape Station

• Used to check RNA quality Protocol
• 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

• 167 had pigment in the final RNA elution