DNA/RNA extractions from E5 project

Re-Extractions from Porites from each of the four timepoints

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Extraction Date: February 01, 2022

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
1	January	Porites	POR-266	20200103	2
25	January	Porites	POR-209	20200103	2
111	January	Porites	POR-381	20200106	3
201	January	Porites	POR-76	20200110	1
287	March	Porites	POR-235	20200303	2
415	March	Porites	POR-75	20200305	1
497	Sept	Porites	POR-253	20200910	2
515	Sept	Porites	POR-216	20200910	2
633	Sept	Porites	POR-73	20200911	1
693	Sept	Porites	POR-80	20200911	1
739	November	Porites	POR-70	20201101	1
907	November	Porites	POR-221	20201030	2

Extraction notes

• I looked at each sample, for those samples that were darker (287, 415, 515, 633, 693) I took 100ul of sample and added it to 200ul of new shield
• For samples that were lighter in pigment, (1, 25, 111, 201, 739, 907) 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	201.28
Standard 2	23737.21
1		4.30	4.10	4.20
25		2.60	2.44	2.52
111		3.08	2.90	2.99
201		2.10	2.00	2.05
287		2.08	nd	2.00
415		2.36	2.20	2.28
497		12.8	12.4	12.6
515		2.04	nd	2.00
633		3.72	3.52	3.62
693		2.54	2.42	2.48
739		2.10	nd	2.00
907		8.92	8.74	8.82

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	423.11
Standard 2	10529.05
1		nd	nd	nd
25		nd	nd	nd
111		nd	nd	nd
201		19.4	19.8	19.6
287		13.8	14.6	14.2
415		13.0	13.0	13.0
497		nd	nd	nd
515		14.8	14.6	14.7
633		18.4	18.2	18.3
693		16.8	16.6	16.7
739		22.4	22.2	22.3
907		nd	nd	nd

Tape Station

• Tape station broken!

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

• NA