Soft and Hard Homogenization Extractions
Soft and Hard Homogenization DNA/RNA Extractions #417-456
20190903 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.
Ran out of wash buffer during RNA extractions and was only able to complete the full RNA extraction for samples #418, 419, 421, and 422.
Qubit used to check DNA and RNA quantity (ng/μl).
TapeStation used to check RNA quality.
DNA Standard 1: 207.83 ng/μl
DNA Standard 2: 22,361.36 ng/μl
RNA Standard 1: 389.41 ng/μl
RNA Standard 2: 9,965.77 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 |
|---|---|---|---|---|---|---|---|---|---|---|---|
| 20180922 | Montipora | 1548 | 417 | soft | 30.2 | 30 | 30.1 | NA | NA | NA | NA |
| 20180922 | Montipora | 1548 | 418 | hard | 12.5 | 12.4 | 12.45 | 17.2 | 17 | 17.1 | NA |
| 20180922 | Montipora | 1628 | 419 | soft | 35.6 | 35.4 | 35.5 | 46 | 46 | 46 | 8.9 |
| 20180922 | Montipora | 1628 | 420 | hard | 21.2 | 21.2 | 21.2 | NA | NA | NA | NA |
| 20180922 | Pocillopora | 1642 | 421 | soft | 46 | 46 | 46 | 105 | 105 | 105 | 8.5 |
| 20180922 | Pocillopora | 1642 | 422 | hard | 40 | 39.8 | 39.9 | 61.2 | 61.2 | 61.2 | NA |
| 20180922 | Pocillopora | 2750 | 423 | soft | 64.2 | 64 | 64.1 | NA | NA | NA | NA |
| 20180922 | Pocillopora | 2750 | 424 | hard | 44.2 | 44 | 44.1 | NA | NA | NA | NA |
| 20180923 | Montipora | 1449 | 425 | soft | 18.7 | 18.6 | 18.65 | NA | NA | NA | NA |
| 20180923 | Montipora | 1449 | 426 | hard | 15.3 | 15.3 | 15.3 | NA | NA | NA | NA |
| 20180923 | Pocillopora | 1563 | 427 | soft | 21.6 | 21.4 | 21.5 | NA | NA | NA | NA |
| 20180923 | Pocillopora | 1563 | 428 | hard | 24.8 | 24.8 | 24.8 | NA | NA | NA | NA |
| 20180923 | Montipora | 1997 | 429 | soft | 18.2 | 18.1 | 18.15 | NA | NA | NA | NA |
| 20180923 | Montipora | 1997 | 430 | hard | 10.5 | 10.5 | 10.5 | NA | NA | NA | NA |
| 20180923 | Montipora | 1694 | 431 | soft | 15.1 | 15 | 15.05 | NA | NA | NA | NA |
| 20180923 | Montipora | 1694 | 432 | hard | 13.2 | 13.2 | 13.2 | NA | NA | NA | NA |
| 20180923 | Pocillopora | 2212 | 433 | soft | 47.6 | 47.4 | 47.5 | NA | NA | NA | NA |
| 20180923 | Pocillopora | 2212 | 434 | hard | 24.2 | 24 | 24.1 | NA | NA | NA | NA |
| 20180923 | Pocillopora | 1707 | 435 | soft | 30.8 | 30.8 | 30.8 | NA | NA | NA | NA |
| 20180923 | Pocillopora | 1707 | 436 | hard | 35.6 | 35.6 | 35.6 | NA | NA | NA | NA |
Link to 20190903 TapeStation report, Extractions #417-436
20190904 E.S. Extraction
Coral fragments #1548 and #1628 re-extracted because not enough wash buffer during yesterday’s extractions to complete the set. See 20190903 description for links to protocols used.
| 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 |
|---|---|---|---|---|---|---|---|---|---|---|---|
| 20180922 | Montipora | 1548 | 437 | soft | 23.2 | 23 | 23.1 | 24.2 | 24.2 | 24.2 | 8 |
| 20180922 | Montipora | 1548 | 438 | hard | 16.9 | 16.9 | 16.9 | 19.8 | 19.8 | 19.8 | NA |
| 20180922 | Montipora | 1628 | 439 | soft | 17 | 17 | 17 | 28.2 | 28.2 | 28.2 | 8.7 |
| 20180922 | Montipora | 1628 | 440 | hard | 21.8 | 21.6 | 21.7 | 24 | 24 | 24 | NA |
DNA Standard 1: 207.83 ng/μl
DNA Standard 2: 22,361.36 ng/μl
RNA Standard 1: 389.41 ng/μl
RNA Standard 2: 9,965.77 ng/μl
Link to 20190904 TapeStation report, Extractions #437-440
20190905 E.S. Extraction
The eight coral fragments that were not re-extracted on 20190904 were extracted on 20190905 because not enough wash buffer during 20190903’s extractions to complete the set. See 20190903 description for links to protocols used.
Fragment preparation start 12:49 end 13:09
Soft/Hard homogenization start 13:10 end 13:38
RNA DNA Extraction start 13:39 end 15:45
DNA Standard 1: 189.79 ng/μl
DNA Standard 2: 21,084.91 ng/μl
RNA Standard 1: 399.67 ng/μl
RNA Standard 2: 9,715.01 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 |
|---|---|---|---|---|---|---|---|---|---|---|---|
| 20180923 | Montipora | 1449 | 441 | soft | 27.2 | 27 | 27.1 | 59.6 | 59.6 | 59.6 | 8.7 |
| 20180923 | Montipora | 1449 | 442 | hard | 22.2 | 22.2 | 22.2 | 26.8 | 26.8 | 26.8 | NA |
| 20180922 | Pocillopora | 1642 | 443 | soft | 70 | 70 | 70 | 80.2 | 80.2 | 80.2 | 7.5 |
| 20180922 | Pocillopora | 1642 | 444 | hard | 37.4 | 37.2 | 37.3 | 40 | 40.2 | 40.1 | NA |
| 20180923 | Pocillopora | 1707 | 445 | soft | 96.6 | 96.4 | 96.5 | 77.4 | 77.6 | 77.5 | 8.5 |
| 20180923 | Pocillopora | 1707 | 446 | hard | 35.4 | 35.4 | 35.4 | 42.2 | 42.4 | 42.3 | NA |
| 20180923 | Pocillopora | 1563 | 447 | soft | 42.4 | 42.2 | 42.3 | 39.8 | 39.8 | 39.8 | 8.7 |
| 20180923 | Pocillopora | 1563 | 448 | hard | 24 | 24 | 24 | 33.2 | 33.2 | 33.2 | NA |
| 20180923 | Pocillopora | 2212 | 449 | soft | 34.2 | 34.2 | 34.2 | 54 | 53.8 | 53.9 | 8.1 |
| 20180923 | Pocillopora | 2212 | 450 | hard | 19.3 | 19.3 | 19.3 | 43.6 | 43.6 | 43.6 | NA |
| 20180923 | Montipora | 1694 | 451 | soft | 19.2 | 19.2 | 19.2 | 25 | 25.2 | 25.1 | 9 |
| 20180923 | Montipora | 1694 | 452 | hard | 14.6 | 14.5 | 14.55 | 16.6 | 16.6 | 16.6 | NA |
| 20180923 | Montipora | 1997 | 453 | soft | 23.4 | 23 | 23.2 | ** | ** | ** | 9.1 |
| 20180923 | Montipora | 1997 | 454 | hard | 11.2 | 11.2 | 11.2 | ** | ** | ** | NA |
| 20180922 | Pocillopora | 2750 | 455 | soft | 60.6 | 60.6 | 60.6 | 85.4 | 85.4 | 85.4 | 8.6 |
| 20180922 | Pocillopora | 2750 | 456 | hard | 45.8 | 45.8 | 45.8 | 45.2 | 45 | 45.1 | NA |
Extraction #453 and #454 read “Too Low” but the TapeStation report a 9.1 value for #453. Think about re-qubitting #453 and #454.
Link to 20190905 TapeStation report, Extractions #441-456
