Soft and Hard Homogenization Extractions
Soft and Hard Homogenization DNA/RNA Extractions #457-464
20190924 E.S. DNA/RNA Extractions from Montipora capitata and Pocillopora acuta adult coral fragments from Holobiont Integration Hawaii 2018 project.
Soft and Hard homogenization, and DNA/RNA Extractions followed this protocol. General Zymo Duet DNA/RNA Extraction protocol found here. With the following changes:
- DNA: The first and second elutions were spun down in separate microcentrifuge tubes to compare the quantity and quality between the first and second elution. The first elution had no incubation period (previously 5 minutes) with 10 μl of Tris buffer and the second elution had a 15 minute incubation period (previously 5 minutes) with 100 μl of Tris buffer.
- 10 μl aliquots of DNA and 5 μl aliquots of RNA (instead of 10 μl of each).
M1236 was very dark brown, almost black and smelled bad. Much darker than the rest of the Montipora fragments.
30 mL of Puritz’s ethanol used. Replaced 10.5 mL of Puritz Wash Buffer.
Qubit used to check DNA and RNA quantity (ng/μl).
TapeStation used to check RNA quality.
DNA Standard 1: 131.30 ng/μl
DNA Standard 2: 18,106.18 ng/μl
RNA Standard 1: 400.44 ng/μl
RNA Standard 2: 10,114.91 ng/μl
| Timepoint | Species | Coral ID | Extraction ID | Homogenization | DNA Reading 1 | DNA Reading 2 | Average DNA ng/μl | RNA Reading 1 | RNA Reading 2 | Average RNA ng/μl | RIN |
|---|---|---|---|---|---|---|---|---|---|---|---|
| 20181006 | Pocillopora | 1281 | 457 | soft | 34.2 | 34 | 34.1 | 64.4 | 64.4 | 64.4 | 8.4 |
| 20181006 | Pocillopora | 1281 | 458 | hard | 16.7 | 16.7 | 16.7 | 39.4 | 39.4 | 39.4 | NA |
| 20180922 | Montipora | 1651 | 459 | soft | 24.6 | 24.4 | 24.5 | 56.6 | 56.8 | 56.7 | 9.1 |
| 20180922 | Montipora | 1651 | 460 | hard | 27 | 27 | 27 | 31.2 | 31.4 | 31.3 | NA |
| 20180922 | Montipora | 1236 | 461 | soft | ** | ** | ** | 17.8 | 17.8 | 17.8 | 2.7 |
| 20180922 | Montipora | 1236 | 462 | hard | ** | ** | ** | 13.6 | 13.8 | 13.7 | NA |
| 20180923 | Montipora | 1229 | 463 | soft | 4.56 | 4.48 | 4.52 | 14.2 | 14.4 | 14.3 | 8.9 |
| 20180923 | Montipora | 1229 | 464 | hard | 3.64 | 3.6 | 3.62 | 15.6 | 15.6 | 15.6 | NA |
20190925 E.C. D.C.
Link to 20190925 TapeStation report, Extractions #457-464
