2022-10-31 ENCORE RNA and DNA Extractions
RNA and DNA Extractions for ENCORE Project, October 31, 2022
Protocol Link
Extraction of 2 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
Forgot to take pictures, but samples were Past-C3 and Past-E3. Neither tubes were particularly pigmented.
Notes
Today I tried adding BME (beta-mercaptoethanol) to the lysis buffer, as is directed in Zymo’s Pathogen and Viral Quick RNA kits. They recommend the following procedure:
- Add 500 μl beta-mercaptoethanol (user supplied) per 100 ml Lysis DNA/RNA Buffer, (final 0.5% (v/v)).
- Add 400 µl of Genomic Lysis Buffer to 100 µl of blood, serum, or plasma (4:1) 3.
- Mix completely by vortexing 4-6 seconds, then let stand 5-10 minutes at room temperature.
So, for a test batch I added 15 uL of BME to a tube with 3 mL of the DNA/RNA Lysis Buffer supplied in the kit. I used this as normal in the extraction protocol, but with twice the amount of Lysis buffer added, as is also recommended by the Viral and Pathogen kits. I decided to divide the extractions into two samples, one that was bead-beat and one that just used DNA/RNA shield from the sample tube.
- For the bead-beat samples, I bead-beat a fragment from the tube in 800 uL of DNA/RNA shield, 200 uL of which came from the sample tube (see my last extraction post). I took the supernatant into the extraction as written in that post.
- For the “100 uL + BME” samples I took 100 uL from the sample DNA/RNA shield and diluted with 200 uL of clean DNA/RNA shield.
When the lysis buffer step came, I added 700 uL instead of the usual 345 uL. This meant I had to spin the flow through through multiple times.
Other than the adjustments mentioned above, I followed protocol exactly.
Qubit Results
- Used Broad range dsDNA and RNA Qubit Protocol
- All samples read twice, standard only read once
DNA Standards: 195.90 (S1) & 22394.56 (S2)
| Sample # | DNA_QBIT_1 | DNA_QBIT_2 | DNA_QBIT_AVG |
|---|---|---|---|
| Past-C3, bead-beat + BME | nd | nd | 0 |
| Past-E3, bead-beat + BME | nd | nd | 0 |
| Past-C3, 100 uL + BME | nd | nd | 0 |
| Past-E3, 100 uL + BME | nd | nd | 0 |
RNA Standards: 409.86 (S1) & 9471.14 (S2)
| Sample # | RNA_QBIT_1 | RNA_QBIT_2 | RNA_QBIT_AVG |
|---|---|---|---|
| Past-C3, bead-beat + BME | nd | nd | 0 |
| Past-E3, bead-beat + BME | nd | nd | 0 |
| Past-C3, 100 uL + BME | nd | nd | 0 |
| Past-E3, 100 uL + BME | nd | nd | 0 |
DNA and RNA Quality Check: Gel, using Kristina’s gel protocol at the bottom of this page.
Next steps
Taking a step back from Porites extractions for a bit. We need to brainstorm if we should try a new kit or just leave these behind for now. There is clearly a little bit of DNA and some degraded RNA in the Bead-Beat samples, but the BME did not help with anything clearly. Maybe adding the 200 uL of sample DNA/RNA shield was a mistake and introduced inhibitors? Either way, we will take a break for now to brainstorm.