20201106 DNA RNA Extractions Porites July Bleaching Experiment and WGBS samples
Project: Porites Bleaching 2019
Goal
To test DNA and RNA extractions on adult Porites astreoides samples for the Porites Bleaching 2019 experiment and 2 samples from Hawaii for WGBS using this protocol.
Samples
Today I am using 4 adult P. astreoides samples, 1 PORITES III (from Hawaii) and 1 MEANDRINA III (from Hawaii) samples that were snap frozen in the field. All of these samples are whole fragments that are stored at -80°C.
Vial # | Timepoint | Coral ID |
---|---|---|
19 | - | MEA III |
20 | - | POR III |
21 | A2 | R29 |
22 | A2 | R11 |
23 | A4 | R20 |
24 | A4 | R28 |
Sample Preparation and Digestion
- Add 0.25 mL of glass beads (0.5mm) and add 500 μl of RNA/DNA shield into each empty centrifuge tube.
- Take samples out from -80 °C on dry ice.
- Using sterile clippers, add 0.25mm of tissue into the centrifuge tube with beads and RNA/DNA shield.
- Vortex at max speed for 2 minutes.
- Only vortexed vial 19 for 45 seconds
- Remove 400 μl of the supernatant and transfer to a new centrifuge tube.
- Centrifuge at 9000 rcf for 3 minutes.
- Transfer 300 μl supernatant to a new centrifuge tube and discard the pellet.
- 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.
- Vortex and spin down.
- Add 345 μl of lysis buffer.
- Continue with DNA and RNA extraction protocol
Quantify Results
Qubit
To test DNA quantity: Qubit
I used DNA and RNA broad range.
DNA (ng/uL) | RNA (ng/uL) | |
---|---|---|
Standard 1 | 193.17 | 391.08 |
Standard 2 | 22259.37 | 8987.79 |
19 | 98.2 | 95.0 |
20 | 16.0 | 30.4 |
21 | 22.4 | 29.2 |
22 | 21.4 | 31.4 |
23 | 10.7 | 15.6 |
24 | 11.0 | 28.0 |
Gel
To test DNA quality: Gel Electrophoresis
TapeStation
Summary:
Sample #19:
Sample #20:
Sample #21:
Sample #22:
Sample #23:
Sample #24:
Full TapeStation report can be found here
Written on November 6, 2020