DNA/RNA extractions from E5 project

Re-Extractions from Porites from each of the four timepoints

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Extraction Date: February 03, 2022

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
19	January	Porites	POR-224	20200103	2
97	January	Porites	POR-367	20200106	3
149	January	Porites	POR-365	20200106	3
191	January	Porites	POR-71	20200110	1
209	January	Porites	POR-79	20200110	1
353	March	Porites	POR-353	20200304	3
475	March	Porites	POR-78	20200305	1
543	Sept	Porites	POR-224	20200910	2
591	Sept	Porites	POR-355	20200908	3
635	Sept	Porites	POR-79	20200911	1
843	November	Porites	POR-381	20201031	3
867	November	Porites	POR-209	20201030	2

Extraction notes

• I looked at each sample, for those samples that were darker (209, 353, 475, and 843) I took 100ul of sample and added it to 200ul of new shield
• For samples that were lighter in pigment, (19, 97, 149, 191, 543, 591, and 867) I took 200ul of sample and added to 100ul of new shield
• For samples that were extremely dark (635) I only took 50ul of sample and added it 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	196.20
Standard 2	20535.66
19		nd	nd	nd
97		nd	nd	nd
149		nd	nd	nd
191		nd	nd	nd
209		nd	nd	nd
353		nd	nd	nd
475		nd	nd	nd
543		18.4	18.0	18.2
591		nd	nd	nd
635		nd	nd	nd
843		11.2	10.9	11.05
867		6.26	5.96	6.11

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	423.64
Standard 2	10284.26
19		nd	nd	nd
97		nd	nd	nd
149		14.4	14.4	14.4
191		11.4	11.4	11.4
209		12.4	12.6	12.5
353		nd	nd	nd
475		nd	nd	nd
543		nd	nd	nd
591		11.4	11.2	11.3
635		nd	nd	nd
843		nd	nd	nd
867		nd	nd	nd

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

• These samples were QC-ed with the samples from Feb 8, 2022. Those standards were made incorrectly so I just used the same DNA and RNA standards from that date for these
• 635 still had pigment in the final RNA elution