Soft and Hard Homogenization Extractions
Soft and Hard Homogenization DNA/RNA Extractions #283-302
20190806 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).
Coral fragments were randomly chosen from different timepoint bags.
Fragment preparation start 13:05 end 13:34
Soft/Hard homogenization start 13:35 end 14:25
RNA DNA Extraction start 14:26 end 17:05
Qubit used to check DNA and RNA quantity (ng/μl).
TapeStation used to check RNA quality.
DNA Standard 1: 199.17 ng/μl
DNA Standard 2: 21,160.02 ng/μl
RNA Standard 1: 396.79 ng/μl
RNA Standard 2: 10,606.91 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 | Montipora | 1722 | 283 | soft | 5.34 | 5.1 | 5.22 | 16.2 | 16 | 16.1 | 8.7 |
| 20181006 | Montipora | 1722 | 284 | hard | 6.78 | 6.8 | 6.79 | 13.8 | 13.6 | 13.7 | 8.4 |
| 20181006 | Montipora | 1455 | 285 | soft | 6.16 | 6.1 | 6.13 | 15.8 | 15.8 | 15.8 | 9.1 |
| 20181006 | Montipora | 1455 | 286 | hard | 5.2 | 5.14 | 5.17 | ** | ** | ** | ** |
| 20181006 | Montipora | 1645 | 287 | soft | 8.94 | 8.88 | 8.91 | 21 | 21 | 21 | 8.6 |
| 20181006 | Montipora | 1645 | 288 | hard | 10.3 | 10.2 | 10.25 | 13.2 | 12.8 | 13 | 9 |
| 20180922 | Montipora | 1269 | 289 | soft | 8.96 | 8.9 | 8.93 | 18 | 18 | 18 | 8.6 |
| 20180922 | Montipora | 1269 | 290 | hard | 11.1 | 11 | 11.05 | 16.2 | 16.2 | 16.2 | 9 |
| 20180922 | Montipora | 1826 | 291 | soft | 18.4 | 18.3 | 18.35 | 25.8 | 25.8 | 25.8 | 8.9 |
| 20180922 | Montipora | 1826 | 292 | hard | 19.1 | 18.9 | 19 | 16.8 | 17 | 16.9 | 9 |
| 20181006 | Pocillopora | 1728 | 293 | soft | 108 | 108 | 108 | 78 | 77.8 | 77.9 | 8.1 |
| 20181006 | Pocillopora | 1728 | 294 | hard | 48.2 | 48 | 48.1 | 47.4 | 47.4 | 47.4 | 8.2 |
| 20180922 | Montipora | 1544 | 295 | soft | 14.9 | 14.8 | 14.85 | 22.4 | 22.2 | 22.3 | 8.8 |
| 20180922 | Montipora | 1544 | 296 | hard | 15.6 | 15.5 | 15.55 | 17 | 16.8 | 16.9 | 8.9 |
| 20181006 | Montipora | 1223 | 297 | soft | 11.9 | 11.9 | 11.9 | 14.4 | 14.4 | 14.4 | 9.2 |
| 20181006 | Montipora | 1223 | 298 | hard | 6.02 | 5.96 | 5.99 | 11.8 | 12 | 11.9 | ** |
| 20180929 | Montipora | 1611 | 299 | soft | 11.5 | 11.4 | 11.45 | 20.4 | 20.4 | 20.4 | 8.8 |
| 20180929 | Montipora | 1611 | 300 | hard | 5.66 | 5.62 | 5.64 | 18.4 | 18.6 | 18.5 | 8.9 |
| 20181006 | Montipora | 2875 | 301 | soft | 29 | 29 | 29 | 26.4 | 26.2 | 26.3 | 8.7 |
| 20181006 | Montipora | 2875 | 302 | hard | 11.4 | 11.3 | 11.35 | 17.6 | 18.2 | 17.9 | 8.7 |
Not enough tapestation tips, only the first 7 samples ran.
Link to 20190806 TapeStation report, Extractions #283-289
20190809 edit:
Link to 20190809 TapeStation report, Extractions #290-342
| Coral ID | Extraction ID | RIN |
|---|---|---|
| 1269 | 290 | 9 |
| 1826 | 291 | 8.9 |
| 1826 | 292 | 9 |
| 1728 | 293 | 8.1 |
| 1728 | 294 | 8.2 |
| 1544 | 295 | 8.8 |
| 1544 | 296 | 8.9 |
| 1223 | 297 | 9.2 |
| 1223 | 298 | ** |
| 1611 | 299 | 8.8 |
| 1611 | 300 | 8.9 |
| 2875 | 301 | NA |
| 2875 | 302 | 8.7 |
| 2860 | 303 | 9.1 |
| 2860 | 304 | 9 |
| 1126 | 305 | 8.7 |
| 1126 | 306 | 8.8 |
| 2204 | 307 | 8.6 |
| 2204 | 308 | 9.1 |
| 1744 | 309 | 8.4 |
| 1744 | 310 | 8.2 |
| 1427 | 311 | 8.8 |
| 1427 | 312 | 8.5 |
| 1090 | 313 | NA |
| 1090 | 314 | 8.1 |
| 1323 | 315 | 9.2 |
| 1323 | 316 | ** |
| 1487 | 317 | 7.4 |
| 1487 | 318 | 7.3 |
| 2878 | 319 | 8.2 |
| 2878 | 320 | 7.9 |
| 2414 | 321 | 8.6 |
| 2414 | 322 | 8.2 |
| 2409 | 323 | 8.2 |
| 2409 | 324 | 7.8 |
| 1047 | 325 | NA |
| 1047 | 326 | 7.8 |
| 1595 | 327 | 8.5 |
| 1595 | 328 | 8.6 |
| 1499 | 329 | 9 |
| 1499 | 330 | 8.7 |
| 2068 | 331 | 8.7 |
| 2068 | 332 | ** |
Extractions #: 301, 313, 325, and 337 did not run because not enough TapeStation screentapes.
20190812 edit:
TapeStation ran for Extraction ID #s: 301, 313, 325, and 337.
Link to 20190812 TapeStation report, Extractions #301, 313, 325, 337