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 18, 2021

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
103	January	Pocillopora	POC-391	20200106	3
117	January	Pocillopora	POC-386	20200106	3
143	January	Acropora	ACR-379	20200106	3
391	March	Pocillopora	POC-55	20200305	1
401	March	Pocillopora	POC-53	20200305	1
461	March	Porites	POR-381	20200304	3
693	Sept	Porites	POR-80	20200911	1
719	November	Acropora	ACR-187	20201101	1
721	November	Pocillopora	POC-45	20201101	1
831	November	Porites	POR-349	20201031	3
869	November	Pocillopora	POC-238	20201030	2
891	November	Pocillopora	POC-259	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	192.06
Standard 2	21666.79
103		18.7	17.3	18.0
117		29.0	28.6	28.8
143		34.4	34.0	34.2
391		41.4	41.0	41.2
401		30.8	30.8	30.8
461		4.02	4.04	4.03
693		2.20	2.12	2.16
719		77.4	77.4	77.4
721		36.6	37.0	36.8
831		nd	nd	nd
869		2.96	2.88	2.92
891		60.4	59.6	60.0

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	423.16
Standard 2	10693.18
103		14.2	14.2	14.2
117		22.8	22.8	22.8
143		nd	nd	nd
391		29.8	30.0	29.9
401		nd	nd	nd
461		nd	nd	nd
693		nd	nd	nd
719		11.0	10.8	10.9
721		26.2	26.4	26.3
831		34.8	35.0	34.9
869		27.8	27.0	27.4
891		19.8	19.0	19.4

Tape Station

• Used to check RNA quality Protocol
• Did not tape station #461 and 693
• 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

• 693 and 831 had pigment in the final RNA elution