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 21, 2021

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
231	January	Acropora	ACR-176	20200110	1
237	January	Pocillopora	POC-40	20200110	1
247	January	Porites	POR-74	20200110	1
455	March	Pocillopora	POC-375	20200304	3
467	March	Acropora	ACR-140	20200305	3
475	March	Porites	POR-78	20200305	1
651	Sept	Pocillopora	POC-53	20200911	1
673	Sept	Acropora	ACR-150	20200911	1
677	Sept	Porites	POR-75	20200911	1
789	November	Pocillopora	POC-369	20201031	3
805	November	Pocillopora	POC-373	20201031	3
807	November	Porites	POR-387	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	184.72
Standard 2	18696.43
231		16.5	16.1	17.95
237		28.4	28.2	44.9
247		nd	nd	nd
455		45.4	44.0	44.7
467		27.6	27.4	4.79
475		2.0	nd	2.0
651		60.2	59.8	52.9
673		60.4	60.0	18.05
677		9.4	9.26	16.15
789		45.4	45.2	68.8
805		52.8	52.4	33.2
807		2.64	2.58	2.46

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	364.46
Standard 2	7066.31
231		17.8	18.1	17.95
237		44.6	45.2	44.9
247		2.28	2.42	2.35
455		15.6	15.8	15.7
467		4.64	4.94	4.79
475		22.4	22.6	22.5
651		53.0	52.8	52.9
673		17.8	18.3	18.05
677		16.2	16.1	16.15
789		68.6	67.0	68.8
805		33.2	33.2	33.2
807		2.46	2.46	2.46

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

• 677 some pigment carryover
• 455 in the sample vial was super dark
• There is band on the gDNA gel for 247 even though qubit said none