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 27, 2021

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
119	January	Acropora	ACR-374	20200106	3
123	January	Pocillopora	POC-371	20200106	3
145	January	Porites	POR-338	20200106	3
339	March	Acropora	ACR-364	20200304	3
369	March	Pocillopora	POC-394	20200304	3
397	March	Pocillopora	POC-40	20200305	1
649	Sept	Porites	POR-81	20200911	1
661	Sept	Pocillopora	POC-40	20200911	1
667	Sept	Acropora	ACR-175	20200911	1
803	November	Porites	POR-367	20201031	3
821	November	Pocillopora	POC-359	20201031	3
835	November	Acropora	ACR-368	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	182.74
Standard 2	19551.12
119		18.5	17.5	18.0
123		35.2	34.8	35.0
145		nd	nd	nd
339		18.1	18.1	18.1
369		11.9	11.8	11.85
397		22.2	22.0	22.1
649		2.38	2.28	2.33
661		56.4	55.4	55.9
667		48.4	47.8	48.1
803		2.80	2.84	2.82
821		25.4	23.8	24.6
835		24.2	23.6	23.9

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	348.63
Standard 2	6663.89
119		10.0	10.8	10.4
123		29.8	28.8	29.3
145		nd	nd	nd
339		11.4	11.4	11.4
369		37.0	37.4	37.2
397		45.2	46.2	45.7
649		15.8	16.0	15.9
661		37.2	37.6	37.4
667		29.2	29.8	29.5
803		25.0	25.0	25.0
821		29.2	29.4	29.3
835		nd	nd	nd

Tape Station

• Used to check RNA quality Protocol
• Did not tape station #145 and 835
• 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

• 649 and 803 some pigment carryover
• There is a very light band in gDNA gel for 145 even though the qubit did not detect any