RNA and DNA Extractions for ENCORE Priming Project

Protocol Link and Here

Extraction of 8 samples from ENCORE Priming Project. These samples were collected in Bermuda in June 2024 for the ENCORE Priming project. There are 100 Madracis decactis (MDEC) samples total.

Samples

These samples are apart of day 13 of processing. Samples were randomly selected out of the 100 total being used for Extractions. Samples were either apart of a three phase priming experiment that included the first stage; Priming where the coral were exposed to 3 treatments high temperature (31ºC), low pH (~7.75) and control (27ºC). The second stage; recovery, where they underwent a week recovery period under control conditions (28ºC). The thrid stage was exposure to a stress which was high temperature (32ºC).

Samples MD-5, MD-5-18, MD-4, MD-4-4, MD-4-6, MD-1-19 were extracted 2025-06-05 by FF but qbitted and ran on a gel the next day with the other samples which was extracted by PP as part of molecular training.

Notes

• Followed this protocol exactly.
• RNA and DNA were extracted from frozen samples in 1mL of DNA/RNA shield
• For sample preperation bead beat was used and added to the orginal tube. The sample was then votexed for 1 mintue and spinned downed after.
• Durin the DNA extraction samples were inverted in step 5 not flicked.
• During both extractions samples were eluted in 80uL of Tris or DNase/RNase free water (50uL on the first elution and 30 on the next).

Qubit Results

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

RNA Standards: 427.62 (S1) 8212.13 (S2)

| colony_id | Species | RNA_QBIT_1 | RNA_QBIT_2 | RNA_QBIT_AVG | |———–|—————————|————|————|————–| | MD-4 | Madracis decactis | 56.8 | 58.0 | 57.4 | | MD-4-6 | Madracis decactis | OUT OF RANGE | | | | MD-5 | Madracis decactis | 37.8 | 38.6 | 38.2 | | MD-5-8 | Madracis decactis | OUT OF RANGE | | | | MD-5-18 | Madracis decactis | 14.4 | 13.4 | 13.9 | | MD-4-4 | Madracis decactis | 11.6 | 11.4 | 11.5 | | MD-1-19 | Madracis decactis | OUT OF RANGE | | | MD-2-22 | Madracis decactis | 20.6 | 19.8 | 20.2 | | MD-4-19 | Madracis decactis | 40.8 | 39.8 | 40.3 | | MD-5-23 | Madracis decactis | 12.8 | 12.4 | 12.6|

DNA and RNA Quality Check: Gel, using this protocol

Note: DNA from sample MD-4-6 came out the gel along with samples MD-1-19 and MD-5-8. Sample MD-4-4 ran on a different gel showing a very faint 1 band, gel should be reran with the sample along with the samples that came out of the gel. Only 2 sample were out of range and will be redone by NC with the high sensitivity kit.