DNA/RNA extractions from E5 project

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

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Extraction Date: October 07, 2021

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
159	January	Pocillopora	POC-373	20200106	3
163	January	Porites	POR-340	20200106	3
169	January	Acropora	ACR-343	20200106	3
273	March	Pocillopora	POC-200	20200303	2
275	March	Porites	POR-240	20200303	2
325	March	Pocillopora	POC-248	20200303	2
537	Sept	Pocillopora	POC-255	20200910	2
543	Sept	Porites	POR-224	20200910	2
555	Sept	Pocillopora	POC-238	20200910	2
755	November	Pocillopora	POC-68	20201101	1
769	November	Porites	POR-71	20201101	1
771	November	Acropora	ACR-186	20201101	1

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	187.99
Standard 2	21874.64
159		14.9	14.7	14.8
163		nd	nd	nd
169		19.3	19.0	19.15
273		93.4	91.6	92.5
275		3.20	3.14	3.17
325		156	154	155
537		nd	nd	nd
543		14.8	14.5	14.65
553		3.46	3.34	3.40
755		53.6	52.8	53.2
769		nd	nd	nd
771		69.2	68.4	68.8

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	374.98
Standard 2	6965.01
159		15.6	16.0	15.8
163		20.6	20.8	20.7
169		nd	nd	nd
273		33.2	33.4	33.3
275		11.2	11.8	11.5
325		39.2	39.6	39.4
537		50.2	51.4	50.8
543		nd	nd	nd
553		51.0	51.6	51.3
755		33.8	34.8	34.3
769		15.8	15.8	15.8
771		15.4	16.0	15.7

Tape Station

• Used to check RNA quality Protocol
• Did not tape station # 169 and 543
• 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

• 769 some pigment carryover in RNA.