DNA/RNA extractions from E5 project

Extractions from each of the three coral species from each of the four timepoints

---

Extraction Date: August 31, 2021

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
25	January	Porites	POR-209	20200103	1
57	January	Acropora	ACR-247	20200103	1
63	January	Pocillopora	POC-259	20200103	1
347	March	Acropora	ACR-350	20200304	3
349	March	Pocillopora	POC-372	20200304	3
353	March	Porites	POR-353	20200304	3
499	Sept	Acropora	ACR-241	20200910	Mahana
501	Sept	Pocillopora	POC-207	20200910	Mahana
509	Sept	Porites	POR-221	20200910	Mahana
709	November	Pocillopora	POC-42	20201101	Mahana
713	November	Porites	POR-69	20201101	Manava
715	November	Acropora	ACR-178	20201101	Manava

Extraction notes

• Samples from the second time point had a lot of biomass in relation to the amount to DNA/RNA shield
• ACR and POC samples: pulled out 300ul of shield
• POR samples: pulled out 150ul and then added 150ul of new shield. The thought behind this was to try and limit the amount of inhibitor
• 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
• 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	184.0
Standard 2	202102.32
25		nd	nd	nd
57		17.1	16.7	16.9
63		7.98	7.68	7.83
347		15.2	14.8	15.0
349		12.4	11.9	12.15
353		3.0	2.9	2.95
499		18.7	18.0	18.35
501		12.8	12.9	12.85
509		2.86	2.74	2.8
709		17.3	17.0	17.15
713		2.88	2.86	2.87
715		17.6	17.5	17.55

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	396.96
Standard 2	6998.54
25		nd	nd	nd
57		14.8	14.2	14.5
63		23.6	23.8	23.7
347		nd	nd	nd
349		33.6	33.6	33.6
353		nd	nd	nd
499		14.6	14.6	14.6
501		22.0	20.6	21.3
509		10.2	10.0	10.1
709		39.0	38.8	38.9
713		23.2	22.2	22.7
715		11.8	11.6	11.7

Tape Station

• Used to check RNA quality Protocol
• I did not tape station 25, 347, and 353 because there was no detectable RNA
• 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

• Stil haven’t figured out why Porites only works about half the time.