RNA and DNA Extractions for ENCORE Project, October 03, 2022

Protocol Link

Extraction of 8 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 4 species: Diploria labyrinthiformis (DLAB), Montastraea cavernosa (MCAV), Madracis decactis (MDEC), and Porites astreoides (PAST)

Samples

These samples are part of our “Round A” of processing. We selected 16 samples from the 160 that were representative of all 4 species at the following 4 temperatures: 16 ºC, 26 ºC, 29 ºC, and 36 ºC. Within each species, the four samples selected for “Round A” all originated from different colonies (given by the letter after the species. For example, for sample Dlab-D1, the colony was colony D and the fragment was fragment 1.)

Notes

• Followed protocol exactly. Took 300 uL from each sample tube.

• Sample Past-F4 looked bad at the step where DNA/RNA Lysis buffer was added to the supernatant at step 6. The supernatant from the Proteinase K digestion appeared to be mucous-y and was immiscible with the Lysis buffer. Throughout the RNA extraction, the filter was pigmented and the RNA was pale brown. This might be an example sample to re-extract with only 100 or 150 uL of the DNA/RNA shield from the sample tube.

Qubit Results

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

DNA Standards: 201.70 (S1) & 23899.02 (S2)

colony_id	Species	Temp.Cat	DNA_QBIT_1	DNA_QBIT_2	DNA_QBIT_AVG
Dlab-D1	Diploria labyrinthiformis	29	4.2	4.08	4.14
Dlab-F6	Diploria labyrinthiformis	26	2.2	2.14	2.17
Mcav-B1	Montastraea cavernosa	29	3.7	3.7	3.7
Mcav-C4	Montastraea cavernosa	16	6.86	6.78	6.82
Mdec-A7	Madracis decactis	36	15	14.9	14.95
Mdec-C6	Madracis decactis	26	6.24	6.08	6.16
Past-D7	Porites astreoides	36	2.34	2.24	2.29
Past-F4	Porites astreoides	16	3.26	3.14	3.2

RNA Standards: 414.70 (S1) & 9890.95 (S2)

colony_id	Species	Temp.Cat	RNA_QBIT_1	RNA_QBIT_2	RNA_QBIT_AVG
Dlab-D1	Diploria labyrinthiformis	29	12.4	12.2	12.3
Dlab-F6	Diploria labyrinthiformis	26	nd	nd	0
Mcav-B1	Montastraea cavernosa	29	14.8	14.8	14.8
Mcav-C4	Montastraea cavernosa	16	12.2	12.2	12.2
Mdec-A7	Madracis decactis	36	18.4	18.2	18.3
Mdec-C6	Madracis decactis	26	16.8	16.6	16.7
Past-D7	Porites astreoides	36	nd	nd	0
Past-F4	Porites astreoides	16	14.4	14.4	14.4

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

Next steps

I will try to re-extract the two PAST samples, this time taking only 150 uL of the DNA/RNA shield from the sample tube and diluting this with another 150 uL prior to the Proteinase K digestion.