DNA/RNA extractions from E5 project

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

---

Extraction Date: September 3, 2021

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
217	January	Acropora	POC-369	20200110	1
235	January	Porites	POR-341	20200110	1
245	January	Pocillopora	ACR-389	20200110	1
393	March	Pocillopora	POC-43	20200304	3
409	March	Porites	ACR-178	20200304	3
423	March	Acropora	POR-75	20200305	3
547	Sept	Acropora	POR-245	20200910	2
577	Sept	Pocillopora	ACR-244	20200908	2
583	Sept	Porites	POC-205	20200908	2
711	November	Porites	ACR-173	20201101	1
717	November	Acropora	POC-43	20201101	1
737	November	Pocillopora	POR-81	20201101	1

Extraction notes

• 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, 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	247.87
Standard 2	21102.64
217		6.82	6.46	6.64
235		nd	nd	nd
245		3.72	4.14	3.93
393		29.6	29.2	29.4
409		nd	nd	nd
423		17.7	17.7	17.7
547		3.94	4.04	3.99
577		nd	nd	nd
583		14.3	14.6	14.45
711		nd	nd	nd
717		10.8	10.9	10.85
737		13.9	13.6	13.75

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	367.79
Standard 2	6965.03
217		nd	nd	nd
235		17.0	15.8	16.4
245		12.4	12.8	12.6
393		27.2	27.4	27.3
409		27.0	27.2	27.1
423		12.8	13.2	13.0
547		21.4	21.4	21.4
577		10.0	10.0	10.0
583		14.0	13.4	13.7
711		17.4	17.4	17.4
717		nd	nd	nd
737		39.8	40.4	40.1

Tape Station

• Used to check RNA quality Protocol
• I did not tape station 217, and 717 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

• 711 had some pigment after the RNA elution steps
• I need to requbit the DNA from 235, 409, 577 and 711. The gel looks okay, I think I miss pipetted.