DNA/RNA extractions from E5 project

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

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Extraction Date: September 2, 2021

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
105	January	Pocillopora	POC-369	20200103	1
109	January	Porites	POR-341	20200103	1
127	January	Acropora	ACR-389	20200103	1
411	March	Pocillopora	POC-43	20200304	3
413	March	Acropora	ACR-178	20200304	3
415	March	Porites	POR-75	20200304	3
495	Sept	Porites	POR-245	20200910	2
503	Sept	Acropora	ACR-244	20200910	2
507	Sept	Pocillopora	POC-205	20200910	2
775	November	Acropora	ACR-173	20201101	1
781	November	Pocillopora	POC-43	20201101	1
783	November	Porites	POR-81	20201101	1

Extraction notes

• ACR and POC samples: pulled out 300ul of shield
• POR samples: pulled out 150ul and then added 150ul of new shield. The thought behind this was to try and limit the amount of inhibitor
• 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	195.04
Standard 2	19958.11
105		14.0	13.7	13.85
109		nd	nd	nd
127		13.1	13.0	13.05
411		13.1	9.04	11.07
413		19.1	18.9	19.9
415		2.76	2.88	2.82
495		5.6	5.6	5.6
503		16.1	15.9	16.0
507		15.1	14.9	15.0
775		4.04	3.98	4.01
781		18.6	18.3	18.45
783		7.66	7.56	7.61

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	348.62
Standard 2	7004.32
105		25.2	24.8	25.0
109		13.8	13.8	13.8
127		12.4	12.0	12.2
411		18.0	17.6	17.8
413		12.8	12.8	12.2
415		nd	nd	nd
495		19.2	18.8	19.0
503		16.8	16.6	16.7
507		23.8	24.0	23.9
775		nd	nd	nd
781		32.2	31.6	31.9
783		18.8	18.4	18.6

Tape Station

• Used to check RNA quality Protocol
• I did not tape station 415, and 775 because there was no detectable RNA
• 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

• 781 had some pigment after the elution steps