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 05, 2021

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
201	January	Porites	POR-76	20200110	1
249	January	Acropora	ACR-186	20200110	1
265	January	Pocillopora	POC-50	20200110	1
451	March	Porites	POR-384	20200304	3
463	March	Acropora	ACR-396	20200304	3
465	March	Pocillopora	POC-386	20200304	3
671	Sept	Porites	POR-69	20200911	1
689	Sept	Acropora	ACR-173	20200911	1
691	Sept	Pocillopora	POC-55	20200911	1
815	November	Porites	POR-383	20201031	3
819	November	Acropora	ACR-374	20201031	3
825	November	Pocillopora	POC-391	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	197.06
Standard 2	22682.93
201		nd	nd	nd
249		16.6	16.8	16.7
265		12.1	12.2	12.15
451		9.70	9.50	9.60
463		8.10	8.14	8.12
465		31.8	31.6	31.7
671		4.50	4.42	4.46
689		19.8	19.6	19.7
691		20.2	20.0	20.1
815		4.44	4.40	4.42
819		nd	nd	nd
825		3.44	3.38	3.41

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	375.91
Standard 2	6897.10
201		22.2	21.8	22.0
249		nd	nd	nd
265		13.2	13.2	13.2
451		14.6	14.8	14.7
463		15.0	15.6	15.3
465		46.2	45.2	45.7
671		18.0	18.2	18.1
689		15.2	15.0	15.1
691		44.6	44.2	44.4
815		29.8	29.2	29.5
819		21.0	20.8	20.9
825		51.2	50.6	50.9

Tape Station

• Used to check RNA quality Protocol
• I learned that you cannot split a tapestation run over three partially used tapes. This is why it’s split across two files. ~learning~
• Results Link
• 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

• 201 and 815 some pigment carryover. 671 was very pigmented
• gel looks like absolute garbage