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 04, 2021

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
131	January	Porites	POR-242	20200106	3
153	January	Acropora	ACR-234	20200106	3
155	January	Pocillopora	POC-222	20200106	3
281	March	Porites	POR-260	20200303	2
291	March	Acropora	POC-201	20200303	2
299	March	Pocillopora	ACR-347	20200303	2
513	Sept	Porites	POR-72	20200910	2
519	Sept	Acropora	POC-48	20200910	2
521	Sept	Pocillopora	POC-43	20200910	2
793	November	Porites	POR-353	20201031	3
809	November	Acropora	POC-358	20201031	3
813	November	Pocillopora	ACR-393	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	195.48
Standard 2	22164.17
131		nd	nd	nd
153		8.82	8.56	8.69
155		36.8	37.4	37.1
281		7.42	7.14	7.28
291		112	111	111.5
299		108	107	107.5
513		3.66	3.62	3.64
519		113	112	112.5
521		3.82	3.74	3.78
793		2.52	2.46	2.49
809		52.0	51.4	51.7
813		42.2	42.0	42.1

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	368.76
Standard 2	7012.71
131		10.8	11.2	11.0
153		nd	nd	nd
155		42.2	42.4	42.3
281		36.6	36.4	36.5
291		nd	nd	nd
299		37.4	38.2	37.8
513		10.6	11.0	10.8
519		15.8	15.4	15.6
521		33.6	35.0	34.3
793		26.8	27.4	27.1
809		17.0	17.0	17.0
813		48.2	48.0	48.1

Tape Station

• Used to check RNA quality Protocol
• 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

• 793 some pigment carryover
• Both RNA gel and tape station don’t look great
• There is a band in 131 gDNA even though qubit did not detect any