RNA and DNA Extractions for ENCORE Project, October 17, 2022

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

Samples

Continuing on with the rest of the ENCORE extractions.

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.

• Throughout the RNA extraction, the filters of all the PAST samples were pigmented.

• I used a new Tri-track loading dye taken from the Putnam shelf in the -20 ºC freezer in attempt to figure out the source of the banding patterns in the DNA gels. This did not get rid of the banding in the DNA. I also tried moving the gel around on different places on the imager and even turning it upside down and the ladder-like small bands still show up in the DNA lanes. Next, I will try imaging a blank gel and testing out other imagers.

Qubit Results

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

– Qubit results seem very odd, getting nds for DNA with clear bands. Maybe I need to be quicker about measuring the Qubit tubes right after making them. I poured the gel after mixing the Qubit tubes and before measuring them to get the gel running ASAP due to time constraints.

DNA Standards: 189.87 (S1) & 21953.74 (S2)

colony_id	Species	Temp.Cat	DNA_QBIT_1	DNA_QBIT_2	DNA_QBIT_AVG
Dlab-A5	Diploria labyrinthiformis	22	nd	nd	0
Dlab-C6	Diploria labyrinthiformis	26	nd	nd	0
Dlab-F4	Diploria labyrinthiformis	16	nd	nd	0
Mcav-B6	Montastraea cavernosa	26	2.26	2.22	2.24
Mcav-D5	Montastraea cavernosa	22	5.24	5.14	5.19
Mcav-F4	Montastraea cavernosa	16	4.74	4.62	4.68
Mdec-A4	Madracis decactis	16	3.2	3.12	3.16
Mdec-B5	Madracis decactis	22	7.18	7.2	7.19
Mdec-F6	Madracis decactis	26	4.64	4.54	4.59
Past-A4	Porites astreoides	16	3.56	3.48	3.52
Past-E6	Porites astreoides	26	nd	nd	0
Past-F5	Porites astreoides	22	nd	nd	0

RNA Standards: 412.01 (S1) & 9743.89 (S2)

colony_id	Species	Temp.Cat	RNA_QBIT_1	RNA_QBIT_2	RNA_QBIT_AVG
Dlab-A5	Diploria labyrinthiformis	22	nd	nd	0
Dlab-C6	Diploria labyrinthiformis	26	nd	nd	0
Dlab-F4	Diploria labyrinthiformis	16	nd	nd	0
Mcav-B6	Montastraea cavernosa	26	nd	nd	0
Mcav-D5	Montastraea cavernosa	22	nd	nd	0
Mcav-F4	Montastraea cavernosa	16	nd	nd	0
Mdec-A4	Madracis decactis	16	nd	nd	0
Mdec-B5	Madracis decactis	22	14	14.2	14.1
Mdec-F6	Madracis decactis	26	11.6	11.6	11.6
Past-A4	Porites astreoides	16	nd	nd	0
Past-E6	Porites astreoides	26	nd	nd	0
Past-F5	Porites astreoides	22	nd	nd	0

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

Ran the gel for 70 minutes instead of 60 minutes, which seems to help the “smiling” patterns in the ladders in my last 2 extractions.

Next steps

After the 10/05/22 extraction, I was going to test out the following modifications but did not due so today because I wanted to try taking 150 uL of the sample DNA/RNA shield from the PAST samples on a few more samples to try to keep the protocol as consistent across species as possible. Clearly, this did not work, so I plan on trouble-shooting the PAST extractions using the method below on Weds, 10/19/22.

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