RNA and DNA Extractions for ENCORE Project, April 21, 2023

Protocol Link

Extraction of 18 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

The tube for MDEC F2 has the writing MDEC C2 on the side. However, there is also a complete MDEC C2 tube (labelled as such on side and cap) in the box and no other MDEC F2.

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. Eluted all DLAB DNA in RNA in 50 uL instead of 100 uL.

Qubit Results

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

DNA Standards: 192.20 (S1) & 21884.44 (S2)

colony_id	Species	Temp.Cat	DNA_QBIT_1	DNA_QBIT_2	DNA_QBIT_AVG
Dlab-B2	Diploria labyrinthiformis	40	53.2	53.8	53.5
Dlab-B4	Diploria labyrinthiformis	16	3.42	3.52	3.47
Dlab-C3	Diploria labyrinthiformis	33	3.96	3.96	3.96
Dlab-D4	Diploria labyrinthiformis	16	4.9	4.98	4.94
Dlab-D6	Diploria labyrinthiformis	26	2.68	2.74	2.71
Dlab-E3	Diploria labyrinthiformis	33	7.24	7.32	7.28
Dlab-E4	Diploria labyrinthiformis	16	5.04	5.32	5.18
Dlab-E5	Diploria labyrinthiformis	22	3.76	3.78	3.77
Mcav-A4	Montastraea cavernosa	16	11.6	11.7	11.65
Mcav-A5	Montastraea cavernosa	22	10.7	10.8	10.75
Mcav-C1	Montastraea cavernosa	29	9.58	9.72	9.65
Mcav-C7	Montastraea cavernosa	36	29	29.2	29.1
Mcav-E2	Montastraea cavernosa	40	101	101	101
Mcav-F2	Montastraea cavernosa	40	88	87.4	87.7
Mdec-B3	Madracis decactis	33	5.72	5.74	5.73
Mdec-C4	Madracis decactis	16	8.4	8.38	8.39
Mdec-E5	Madracis decactis	22	7.56	7.52	7.54
Mdec-F2	Madracis decactis	40	15.5	15.8	15.65

RNA Standards: 417.49 (S1) & 8557.82 (S2)

colony_id	Species	Temp.Cat	RNA_QBIT_1	RNA_QBIT_2	RNA_QBIT_AVG
Dlab-B2	Diploria labyrinthiformis	40	19	19.8	19.4
Dlab-B4	Diploria labyrinthiformis	16	12.4	12.6	12.5
Dlab-C3	Diploria labyrinthiformis	33	14	14	14
Dlab-D4	Diploria labyrinthiformis	16	12	12	12
Dlab-D6	Diploria labyrinthiformis	26	10.4	10.8	10.6
Dlab-E3	Diploria labyrinthiformis	33	14.8	15.2	15
Dlab-E4	Diploria labyrinthiformis	16	10.8	10.8	10.8
Dlab-E5	Diploria labyrinthiformis	22	14.4	14.4	14.4
Mcav-A4	Montastraea cavernosa	16	24.2	24	24.1
Mcav-A5	Montastraea cavernosa	22	10.8	11	10.9
Mcav-C1	Montastraea cavernosa	29	18.6	18.4	18.5
Mcav-C7	Montastraea cavernosa	36	17	17.2	17.1
Mcav-E2	Montastraea cavernosa	40	27.6	27.2	27.4
Mcav-F2	Montastraea cavernosa	40	23.6	24.6	24.1
Mdec-B3	Madracis decactis	33	14.4	14.4	14.4
Mdec-C4	Madracis decactis	16	25.8	25.4	25.6
Mdec-E5	Madracis decactis	22	28.4	28	28.2
Mdec-F2	Madracis decactis	40	28.4	27.8	28.1

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! 34 + any re-extractions to go! Hoping to finish by the end of the semester.

Still holding off on the Porites astreoides extractions until we can find a suitable protocol.