DNA/RNA extractions from E5 project

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

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Extraction Date: September 24, 2021

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
135	January	Pocillopora	POC-378	20200106	3
141	January	Acropora	ACR-368	20200106	3
149	January	Porites	POR-365	20200106	3
289	March	Pocillopora	POC-205	20200303	2
295	March	Acropora	ACR-229	20200303	2
297	March	Porites	POR-245	20200303	2
559	Sept	Pocillopora	POC-248	20200910	2
623	Sept	Pocillopora	POC-386	20200908	3
625	Sept	Porites	POR-287	20200908	3
829	November	Pocillopora	POC-394	20201031	3
833	November	Acropora	ACR-390	20201031	3
837	November	Porites	POR-362	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	195.01
Standard 2	22598.84
135		26.4	25.2	25.8
141		10.1	10.6	10.35
149		nd	nd	nd
289		113	113	113
295		34.6	34.4	34.5
297		2.72	2.64	2.68
559		19.6	19.7	19.65
623		40.8	41.0	40.9
625		nd	nd	nd
829		21.6	21.6	21.6
833		40.8	40.4	40.6
837		5.68	5.76	5.72

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	352.26
Standard 2	6899.11
135		25.0	25.4	25.2
141		nd	nd	nd
149		11.6	nd?	11.6
289		40.4	38.2	39.3
295		11.0	nd?	11.0
297		23.6	21.0	22.3
559		64.4	61.2	62.8
623		36.0	33.6	34.8
625		12.4	10.2	11.3
829		51.4	48.6	50.0
833		17.0	15.2	16.1
837		31.2	29.0	30.1

Tape Station

• Used to check RNA quality Protocol
• Tape station every sample, because I did not trust the qubit and those funky readings
• 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

• 297 and 625 some pigment carryover
• no clue what was going on with the qubit, gel and tape station look okay
• Qubit did not detect any gDNA in 141 and 625, but there are bands on the gel