Soft and Hard Homogenization Extractions
20191009 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).
DNA 400 μl Wash Buffer step was centrifuged for 30 seconds instead of 2 minutes.
Bleached fragments: P1416 (HTHC), P1596 (HTAC), P2185 (HTHC)
Qubit used to check DNA and RNA quantity (ng/μl).
TapeStation used to check RNA quality.
DNA Standard 1: 149.92 ng/μl
DNA Standard 2: 20,432.03 ng/μl
RNA Standard 1: 396.96 ng/μl
RNA Standard 2: 9,194.56 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 |
|---|---|---|---|---|---|---|---|---|---|---|---|
| 20181215 | Montipora | 2756 | 495 | soft | 29 | 29 | 29 | 37.4 | 37.4 | 37.4 | 9.2 |
| 20181215 | Montipora | 2756 | 496 | hard | 9 | 8.96 | 8.98 | 15.2 | 15.2 | 15.2 | NA |
| 20181215 | Pocillopora | 1416 | 497 | soft | 16.8 | 16.7 | 16.75 | 61.2 | 61.2 | 61.2 | 9 |
| 20181215 | Pocillopora | 1416 | 498 | hard | 6.98 | 6.94 | 6.96 | 22.6 | 22.8 | 22.7 | NA |
| 20181215 | Pocillopora | 2185 | 499 | soft | 26.8 | 26.8 | 26.8 | 49.2 | 49.2 | 49.2 | 9.1 |
| 20181215 | Pocillopora | 2185 | 500 | hard | 15.9 | 15.9 | 15.9 | 28.2 | 28.4 | 28.3 | NA |
| 20181215 | Montipora | 1270 | 501 | soft | 42 | 42 | 42 | 39.2 | 39.4 | 39.3 | 8.9 |
| 20181215 | Montipora | 1270 | 502 | hard | 24.8 | 24.6 | 24.7 | 24.2 | 24.2 | 24.2 | NA |
| 20181215 | Pocillopora | 1596 | 503 | soft | 27.8 | 27.8 | 27.8 | 69.8 | 70 | 69.9 | 9.5 |
| 20181215 | Pocillopora | 1596 | 504 | hard | 13.1 | 13.1 | 13.1 | 44.4 | 44.6 | 44.5 | NA |
| 20190112 | Montipora | 1452 | 505 | soft | 32.6 | 32.6 | 32.6 | 35 | 35 | 35 | 9.2 |
| 20190112 | Montipora | 1452 | 506 | hard | 22.8 | 22.8 | 22.8 | ** | ** | ** | NA |
| 20190112 | Montipora | 2190 | 507 | soft | 48 | 47.8 | 47.9 | 12.4 | 12.4 | 12.4 | 9.1 |
| 20190112 | Montipora | 2190 | 508 | hard | 20.4 | 20.4 | 20.4 | 23.6 | 23.8 | 23.7 | NA |
| 20190112 | Montipora | 1140 | 509 | soft | 23.8 | 23.6 | 23.7 | 20.4 | 20.6 | 20.5 | 9.3 |
| 20190112 | Montipora | 1140 | 510 | hard | 14.5 | 14.5 | 14.5 | 15.4 | 15.6 | 15.5 | NA |
| 20190112 | Montipora | 1632 | 511 | soft | 32.4 | 32.4 | 32.4 | 23.2 | 23.4 | 23.3 | 9.4 |
| 20190112 | Montipora | 1632 | 512 | hard | 11.1 | 11.1 | 11.1 | 20.8 | 20.8 | 20.8 | NA |
| 20190112 | Montipora | 2990 | 513 | soft | 10.8 | 10.8 | 10.8 | 17.2 | 17.4 | 17.3 | 8.9 |
| 20190112 | Montipora | 2990 | 514 | hard | 15.7 | 15.7 | 15.7 | 14 | 14 | 14 | NA |
Link to 20191009 TapeStation report, Extractions #495-514
Written on October 9, 2019