2022-10-05 ENCORE RNA and DNA Extractions

RNA and DNA Extractions for ENCORE Project, October 05, 2022

Extraction of 10 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.) Today, I extracted the remaining 8/16 after the 2022-10-03 extraction as well as re-extracting the two Porites astreoides (PAST) samples from that date that were unsuccessfully extracted (Past-D7 and Past-F4).

22022-10-05-tubes.JPG 2022-10-05-caps.JPG

Notes

  • Followed protocol exactly. Took 300 uL from the sample tubes for all species except for Porites astreoides (PAST). For Porites astreoides (PAST), I took 150 uL of the DNA/RNA shield from the sample tube and added 150 uL of clean DNA/RNA shield before proceeding with the extraction as written.

  • I had to centrifuge the PAST samples Past-A1 and Past-B6 multiple times to successfully pellet the debris after the Proteinase K digestion. I found that using a P200 pipette set to 200 uL was the easiest way to remove the supernatant without disturbing the very loose pellets from the PAST samples.
  • Throughout the RNA extraction, the filters of all the PAST samples were pigmented. Past-A1 had the most pigmented RNA filter column. At the end of the extractio the RNA from samples Past-A1 and Past-F4 were pale brown.

Qubit Results

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

DNA Standards: 198.66 (S1) & 24171.04 (S2)

colony_id Species Temp.Cat DNA_QBIT_1 DNA_QBIT_2 DNA_QBIT_AVG
Dlab-A4 Diploria labyrinthiformis 16 5.28 5.08 5.18
Dlab-C7 Diploria labyrinthiformis 36 10.1 9.92 10.01
Mcav-E6 Montastraea cavernosa 26 4.96 4.8 4.88
Mcav-F7 Montastraea cavernosa 36 12.9 12.8 12.85
Mdec-D4 Madracis decactis 16 5 4.78 4.89
Mdec-E1 Madracis decactis 29 8.06 8.02 8.04
Past-A1 Porites astreoides 29 nd nd 0
Past-B6 Porites astreoides 26 nd nd 0
Past-D7 Porites astreoides 36 nd nd 0
Past-F4 Porites astreoides 16 nd nd 0

RNA Standards: 412.67 (S1) & 9635.27 (S2)

colony_id Species Temp.Cat RNA_QBIT_1 RNA_QBIT_2 RNA_QBIT_AVG
Dlab-A4 Diploria labyrinthiformis 16 13.6 13.4 13.5
Dlab-C7 Diploria labyrinthiformis 36 11.2 10.2 10.7
Mcav-E6 Montastraea cavernosa 26 11.8 11.4 11.6
Mcav-F7 Montastraea cavernosa 36 10.6 10.4 10.5
Mdec-D4 Madracis decactis 16 12.8 12.6 12.7
Mdec-E1 Madracis decactis 29 22.2 21.8 22
Past-A1 Porites astreoides 29 12.4 12 12.2
Past-B6 Porites astreoides 26 nd nd 0
Past-D7 Porites astreoides 36 nd nd 0
Past-F4 Porites astreoides 16 nd nd 0

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

2022-10-05-gel.JPG

Next steps

I will talk to Hollie and Kristina about next steps for the PAST samples. Currently, I am thinking of bead beating the fragments in the tube in a bead tube with fresh DNA/RNA shield following Kevin Wong’s protocol here:

Sample Preparation and Digestion

  1. Add 0.25 mL of glass beads (0.5mm) and add 500 μl of RNA/DNA shield into each empty centrifuge tube.
  2. Take samples out from -80 °C on dry ice.
  3. Using sterile clippers, add 0.25mm of tissue into the centrifuge tube with beads and RNA/DNA shield.
  4. Vortex at max speed for 2 minutes.
  5. Remove 400 μl of the supernatant and transfer to a new centrifuge tube.
  6. Centrifuge at 9000 rcf for 3 minutes.
  7. Transfer 300 μl supernatant to a new centrifuge tube and discard the pellet.
  8. Add 30 μl of Proteinase K digestion buffer (10:1 ratio of sample:digestion buffer), and 15 μl of Proteinase K (2:1 ratio of digestion buffer:Proteinase K) to each sample.
  9. Vortex and spin down.
  10. Add 345 μl of lysis buffer.
  11. Continue with DNA and RNA extraction protocol
Written on October 5, 2022