2023-02-16 ENCORE RNA and DNA Extractions
RNA and DNA Extractions for ENCORE Project, February 16, 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.
- Concentrations were generally pretty low for the RNA today. The lab AC was not on and it got up to 80ºC in the lab and I think this negatively affected RNA quality. Some of these will need to be redone.
Qubit Results
- Used Broad range dsDNA and RNA Qubit Protocol
- All samples read twice, standard only read once
DNA Standards: 167.18 (S1) & 16640.38 (S2)
| colony_id | Species | Temp.Cat | DNA_QBIT_1 | DNA_QBIT_2 | DNA_QBIT_AVG |
|---|---|---|---|---|---|
| Dlab-B1 | Diploria labyrinthiformis | 29 | nd | nd | 0 |
| Dlab-C2 | Diploria labyrinthiformis | 40 | 10.4 | 10.5 | 10.45 |
| Dlab-E7 | Diploria labyrinthiformis | 36 | 16.2 | 16.4 | 16.3 |
| Dlab-F3 | Diploria labyrinthiformis | 33 | 2 | 2 | 2 |
| Mcav-A3 | Montastraea cavernosa | 33 | 7.2 | 7.26 | 7.23 |
| Mcav-A7 | Montastraea cavernosa | 36 | 51 | 51.4 | 51.2 |
| Mcav-C2 | Montastraea cavernosa | 40 | 39.6 | 39.8 | 39.7 |
| Mcav-D4 | Montastraea cavernosa | 16 | 5.64 | 5.68 | 5.66 |
| Mdec-C2 | Madracis decactis | 40 | 18.5 | 18.5 | 18.5 |
| Mdec-D7 | Madracis decactis | 36 | 52 | 52 | 52 |
| Mdec-F7 | Madracis decactis | 36 | 23.2 | 23 | 23.1 |
| Fire Coral S10 | Millepora sp. | NA | 10.7 | 10.7 | 10.7 |
RNA Standards: 385.96 (S1) & 8014.30 (S2)
| colony_id | Species | Temp.Cat | RNA_QBIT_1 | RNA_QBIT_2 | RNA_QBIT_AVG |
|---|---|---|---|---|---|
| Dlab-B1 | Diploria labyrinthiformis | 29 | 12 | 11.8 | 11.9 |
| Dlab-C2 | Diploria labyrinthiformis | 40 | 17.2 | 17.4 | 17.3 |
| Dlab-E7 | Diploria labyrinthiformis | 36 | 17.8 | 17.8 | 17.8 |
| Dlab-F3 | Diploria labyrinthiformis | 33 | nd | nd | 0 |
| Mcav-A3 | Montastraea cavernosa | 33 | 17 | 17.4 | 17.2 |
| Mcav-A7 | Montastraea cavernosa | 36 | 23.2 | 23.8 | 23.5 |
| Mcav-C2 | Montastraea cavernosa | 40 | 14.8 | 15 | 14.9 |
| Mcav-D4 | Montastraea cavernosa | 16 | 16.6 | 17.2 | 16.9 |
| Mdec-C2 | Madracis decactis | 40 | 33.2 | 32.8 | 33 |
| Mdec-D7 | Madracis decactis | 36 | 37.8 | 37.4 | 37.6 |
| Mdec-F7 | Madracis decactis | 36 | 10.6 | 0 | 5.3 |
| Fire Coral S10 | Millepora sp. | NA | nd | nd | 0 |
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.
40 ºC samples seemed to work and don’t look any worst on the gel so I will continue to extract these as well and randomly add them back into the extraction order.
Concentrations were generally pretty low for the RNA today. The lab AC was not on and it got up to 80ºC in the lab and I think this negatively affected RNA quality. Some of these will need to be redone.
The fire coral sample did not work, this is a sample from this series of extraction and we wanted to test RNA extraction for transcriptome for genome annotation. However, the sample was kept in DNA/RNA shield in the fridge and there was very little input material in the tube.