First DNA/RNA extractions

Extractions from each of the three coral species for the 5E project. One vial from each of the study species were used.

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Extraction Date: August 24, 2021

Samples

Tube number	Timepoint	Species	Colony ID	Tube ID	Site
1	January	Porites	POR-266	202000103	2
31	January	Acropora	ACR-267	202000103	2
59	January	Pocillopora	POC-255	202000103	2

Extraction notes

• Half to a third of coral fragment clipped and 500 μl of DNA/RNA shield from sample tube placed in tube with 2 mL 0.5 mm glass beads and 500ul of RNA/DNA shield
• Pocillopora and Acropora were bead beated for one minute while Porites was bead beated for two
• After bead beating the samples, I briefly centrifuged them down to help get the debris to the bottom of the tube and help get rid of some of the foam
• Pulled 300ul of this out and added it to a new 1.5ml tube and used it for the extraction. The volume remaining was placed back into the -80C
• 300ul of tissue homogenate, 15ul of ProK, and 30ul of ProK digestion buffer
• 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	193.51
Standard 2	21113.81
1		6.56	6.24	6.40
31		9.04	8.78	8.91
59		4.22	4.00	4.11

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	360.21
Standard 2	6868.73
1		nd	nd	nd
31		11.2	11.2	11.2
59		24.0	22.8	23.4

Tape Station

• Used to check RNA quality Protocol
• I did not tape station the first sample 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 ~3ul 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

• Need to try and troubleshoot why the Porites extraction didn’t work. There might be some inhibitor that is messing with the extraction of the RNA