RNA and DNA Extractions for ENCORE Project, April 6, 2023

Protocol Link

Extraction of 12 samples from ENCORE Project. These samples were collected in Bermuda in August 2022 for the ENCORE TPC project. There are 160 samples total from the following 3 species: Diploria labyrinthiformis (DLAB), Montastraea cavernosa (MCAV), and Madracis decactis (MDEC) from the following temperatures 16, 22, 26, 29, 33, 36, and 40 ºC

Samples

Continuing on with the rest of the ENCORE extractions.

Notes

• Followed protocol exactly. Took 300 uL from the sample tubes for all species before proceeding with the extraction as written.
• The DLAB DNA and RNA concentrations have been consistenly low but the quality is good. Today I tried out eluting the DLAB RNA in 50 uL instead of 100 uL and it worked great. When doing this I still do the second elution but instead of taking new RNAse free water I take the 50uL that passed through in the first elution and re-apply to the membrane. I will continue to do this for the DLAB RNA in the future and will also do so for the DLAB DNA.

Qubit Results

• Used Broad range dsDNA and RNA Qubit Protocol
• All samples read twice, standard only read once

DNA Standards: 197.48 (S1) & 22870.14 (S2)

colony_id	Species	Temp.Cat	DNA_QBIT_1	DNA_QBIT_2	DNA_QBIT_AVG
Dlab-A2	Diploria labyrinthiformis	40	4.98	4.94	4.96
Dlab-B3	Diploria labyrinthiformis	33	2.84	2.9	2.87
Dlab-C4	Diploria labyrinthiformis	16	0	0	0
Dlab-E6	Diploria labyrinthiformis	26	0	0	0
Dlab-F2	Diploria labyrinthiformis	40	17.2	17.4	17.3
Mcav-C3	Montastraea cavernosa	33	10.9	11	10.95
Mcav-F1	Montastraea cavernosa	29	27.6	27.8	27.7
Mdec-A5	Madracis decactis	22	5.22	5.36	5.29
Mdec-D3	Madracis decactis	33	6.72	6.76	6.74
Mdec-D5	Madracis decactis	22	2.68	2.8	2.74
Mdec-E6	Madracis decactis	26	4.96	5	4.98

RNA Standards: 406.98 (S1) & 8665.72 (S2)

colony_id	Species	Temp.Cat	RNA_QBIT_1	RNA_QBIT_2	RNA_QBIT_AVG
Dlab-A2	Diploria labyrinthiformis	40	17.4	17.4	17.4
Dlab-B3	Diploria labyrinthiformis	33	18.4	18.6	18.5
Dlab-C4	Diploria labyrinthiformis	16	12.6	13.4	13
Dlab-E6	Diploria labyrinthiformis	26	0	0	0
Dlab-F2	Diploria labyrinthiformis	40	17.8	18.2	18
Mcav-C3	Montastraea cavernosa	33	21.6	21.6	21.6
Mcav-F1	Montastraea cavernosa	29	34	34.8	34.4
Mdec-A5	Madracis decactis	22	23.2	23.6	23.4
Mdec-D3	Madracis decactis	33	31	31	31
Mdec-D5	Madracis decactis	22	0	10.2	5.1
Mdec-E6	Madracis decactis	26	22.2	22	22.1

DNA and RNA Quality Check: Gel, using Kristina’s gel protocol at the bottom of this page.

Next steps

Will continue on with the rest of the extractions for these 3 species at these temps! Still holding off on the Porites astreoides extractions until we can find a suitable protocol.