Troubleshooting DNA/RNA extractions from E5 project

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

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Extraction Date: December 02, 2021

Samples

Tube number	Timepoint	Species	Colony ID	Coll date	Site
195	January	Porites	POC-346	20200110	1
199	January	Pocillopora	ACR-396	20200110	1
203	January	Acropora	POR-383	20200110	1
205	January	Pocillopora	POC-257	20200110	1
207	January	Acropora	POR-253	20200110	1
209	January	Porites	POR-266	20200110	1
211	January	Acropora	POC-371	20200110	1
215	January	Porites	POR-362	20200110	1
225	January	Acropora	ACR-210	20200110	1
227	January	Acropora	POC-55	20200110	1
229	January	Acropora	POR-240	20200110	1
10S	N/A	Acropora	10S	N/A	N/A

Extraction notes

• ACR and POC samples: pulled out 300ul of shield
• POR samples: pulled out 150ul of shield and added 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	195.83
Standard 2	28772.93
195		nd	nd	nd
199		13.0	13.0	13.0
203		25.0	25.0	25.0
205		21.0	20.8	20.9
207		NA	NA	NA
209		nd	nd	nd
211		11.4	11.3	11.35
215		nd	nd	nd
225		10.5	10.8	10.65
227		12.7	12.7	12.7
229		19.5	19.4	19.45
10S		14.0	14.0	14.0

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	409.39
Standard 2	13867.92
195		11.0	11.4	11.2
199		11.0	11.0	11.0
203		nd	nd	nd
205		14.2	14.2	14.2
207		NA	NA	NA
209		15.6	15.6	15.6
211		nd	nd	nd
215		nd	nd	nd
225		nd	nd	nd
227		nd	nd	nd
229		nd	nd	nd
10S		nd	nd	nd

Tape Station

• Tape station broke!

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

• Something wrong with the DNA column from #207. Sample was trashed