DNA/RNA extractions from E5 project

Re-Extractions from Porites from three of the timepoints

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Extraction Date: February 17, 2022

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
13	January	Porites	POR-245	20200103	2
15	January	Porites	POR-251	20200103	2
125	January	Porites	POR-355	20200106	3
215	January	Porites	POR-83	20200110	1
243	January	Porites	POR-81	20200110	1
601	Sept	Porites	POR-362	20200909	3
629	Sept	Porites	POR-349	20200908	3
745	November	Porites	POR-75	20201101	1
795	November	Porites	POR-354	20201031	3
901	November	Porites	POR-253	20201030	2
923	November	Porites	POR-240	20201030	2
927	November	Porites	POR-253	20201030	2

Extraction notes

• I looked at each sample, for those samples that were darker (745) I took 100ul of sample and added it to 200ul of new shield
• For samples that were lighter in pigment, (the rest) I took 200ul of sample and added to 100ul 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	202.27
Standard 2	23169.02
13		nd	nd	nd
15		2.12	2.02	2.07
125		nd	nd	nd
215		nd	nd	nd
243		nd	nd	nd
601		nd	nd	nd
629		2.22	2.18	2.20
745		3.88	3.72	3.80
795		2.38	2.34	2.36
901		35.0	34.6	34.8
923		28.2	28.2	28.2
927		19.6	19.3	19.45

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	417.33
Standard 2	10724.70
13		11.8	12.0	11.9
15		nd	nd	nd
125		14.6	15.0	14.8
215		nd	nd	nd
243		11.2	11.8	11.5
601		12.0	12.4	12.2
629		16.6	16.6	16.6
745		16.2	16.4	16.3
795		16.8	16.2	16.6
901		13.8	14.4	14.1
923		15.0	15.2	15.1
927		11.0	10.0	10.5

Tape Station

• Not run

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

• 125, 745, and 795 were pigmented in final RNA elution
• I don’t trust the qubit readings of 901, 923, and 927. There are no bands in the gel