DNA/RNA extractions from E5 project

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

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Extraction Date: September 10, 2021

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
241	January	Acropora	ACR-139	20200110	1
253	January	Pocillopora	POC-41	20200110	1
263	January	Acropora	ACR-187	20200110	1
301	March	Acropora	ACR-237	20200303	2
307	March	Acropora	ACR-258	20200303	2
315	March	Pocillopora	POC-219	20200303	2
525	Sept	Acropora	ACR-218	20200910	2
533	Sept	Pocillopora	POC-200	20200910	2
555	Sept	Pocillopora	POC-222	20200910	2
791	November	Pocillopora	POC-371	20201031	3
801	November	Pocillopora	POC-346	20201031	3
813	November	Pocillopora	POC-378	20201031	3

Extraction notes

• ACR and POC samples: pulled out 300ul of 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	206.46
Standard 2	20887.01
241		21.6	22.2	21.9
253		24.6	24.6	24.6
263		22.0	20.8	21.4
301		40.4	40.4	40.4
307		52.2	52.4	52.3
315		99.4	100.0	99.7
525		70.2	70.6	70.4
533		55.0	55.0	55.0
555		24.4	25.0	24.7
791		53.0	52.6	52.8
801		39.6	39.4	39.5
813		46.8	47.2	47.0

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	371.75
Standard 2	7582.53
241		11.6	11.6	11.6
253		16.8	16.6	16.7
263		nd	nd	nd
301		18.2	18.6	18.4
307		25.8	26.0	25.9
315		78.6	78.4	78.5
525		17.8	17.4	17.6
533		39.6	38.0	38.8
555		26.8	26.6	26.7
791		36.0	36.0	36.0
801		37.2	36.4	36.8
813		49.2	48.2	48.7

Tape Station

• Used to check RNA quality Protocol
• I did not tape station 263 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

• NA