Soft and Hard Homogenization
Soft and Hard Homogenization DNA/RNA Extractions #1-32
20190718 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:
- 1 minute hard homogenization step
Coral fragments were randomly chosen from timepoint bags.
| Timepoint | Coral Fragment ID | Species | Extraction ID | Homogenization |
|---|---|---|---|---|
| 20181117 | 1754 | Montipora capitata | 1 | Soft |
| 20181117 | 1754 | Montipora capitata | 2 | Hard |
| 20180922 | 1775 | Pocillopora acuta | 3 | Soft |
| 20180922 | 1775 | Pocillopora acuta | 4 | Hard |
| 20180922 | 2005 | Pocillopora acuta | 5 | Soft |
| 20180922 | 2005 | Pocillopora acuta | 6 | Hard |
| 20181020 | 2386 | Montipora capitata | 7 | Soft |
| 20181020 | 2386 | Montipora capitata | 8 | Hard |
| 20181103 | 2357 | Pocillopora acuta | 9 | Soft |
| 20181103 | 2357 | Pocillopora acuta | 10 | Hard |
| 20181103 | 1131 | Pocillopora acuta | 11 | Soft |
| 20181103 | 1131 | Pocillopora acuta | 12 | Hard |
| 20180922 | 2026 | Pocillopora acuta | 13 | Soft |
| 20180922 | 2026 | Pocillopora acuta | 14 | Hard |
| 20180922 | 1471 | Pocillopora acuta | 15 | Soft |
| 20180922 | 1471 | Pocillopora acuta | 16 | Hard |
| 20181215 | 1644 | Montipora capitata | 17 | Soft |
| 20181215 | 1644 | Montipora capitata | 18 | Hard |
| 20180922 | 1676 | Pocillopora acuta | 19 | Soft |
| 20180922 | 1676 | Pocillopora acuta | 20 | Hard |
| 20181117 | 1631 | Montipora capitata | 21 | Soft |
| 20181117 | 1631 | Montipora capitata | 22 | Hard |
| 20180922 | 2183 | Montipora capitata | 23 | Soft |
| 20180922 | 2183 | Montipora capitata | 24 | Hard |
| 20181215 | 1103 | Pocillopora acuta | 25 | Soft |
| 20181215 | 1103 | Pocillopora acuta | 26 | Hard |
| 20181103 | 1696 | Pocillopora acuta | 27 | Soft |
| 20181103 | 1696 | Pocillopora acuta | 28 | Hard |
| 20180922 | 1600 | Montipora capitata | 29 | Soft |
| 20180922 | 1600 | Montipora capitata | 30 | Hard |
| 20181103 | 1580 | Montipora capitata | 31 | Soft |
| 20181103 | 1580 | Montipora capitata | 32 | Hard |
The 2 ATAC December timepoint coral fragments (1 Pocillopora, 1 Montipora) will be used for DNA methylation protocol testing.
Extractions #1-16 done together and #17-32 done together.
Extractions #1-16:
Fragment Prep - start 10:00 end 10:42
Soft/Hard homogenization - start 10:43 end 11:22
Extractions - start 11:23 end 14:30
New DNase I Stock made and used for all samples.
Extractions #17-32:
Fragment Prep - start 14:30 end 15:00
Soft/Hard homogenization - start 15:00 end 15:37
Extractions - start 15:37 end 18:25
DNAse I Master Mix was not enough for all samples. New DNase I master mix made for last 2 samples.
Extraction ID #19 has 150 μl final volume.
20190719 E.S.
Gel electrophoresis, Qubit, and TapeStation from extractions #1-32.
Qubit Results:
| DNA | |||
|---|---|---|---|
| Standard 1 | 101.91 | ||
| Standard 2 | 8942.34 | ||
| Reading 1 | Reading 2 | Average | |
| 1 | 28 | 27.6 | 27.8 |
| 2 | 17.1 | 17 | 17.05 |
| 3 | 46.4 | 46 | 46.2 |
| 4 | 37.4 | 37 | 37.2 |
| 5 | 42.6 | 42.4 | 42.5 |
| 6 | 30.4 | 30.2 | 30.3 |
| 7 | 52.2 | 51.8 | 52 |
| 8 | 36.2 | 36 | 36.1 |
| 9 | 57.4 | 57 | 57.2 |
| 10 | 62 | 61.6 | 61.8 |
| 11 | 35.2 | 35 | 35.1 |
| 12 | 30.6 | 30.4 | 30.5 |
| 13 | 12 | 11.9 | 11.95 |
| 14 | 14.9 | 14.8 | 14.85 |
| 15 | 55 | 54.6 | 54.8 |
| 16 | 41.4 | 41 | 41.2 |
| 17 | 42.8 | 42.4 | 42.6 |
| 18 | 34.2 | 34 | 34.1 |
| 19 | 28.2 | 27.8 | 28 |
| 20 | 24 | 23 | 23.5 |
| 21 | 20.4 | 20.4 | 20.4 |
| 22 | 20.2 | 20.2 | 20.2 |
| 23 | 16.9 | 16.8 | 16.85 |
| 24 | 11.7 | 11.5 | 11.6 |
| 25 | 56.8 | 56.4 | 56.6 |
| 26 | 51 | 50.8 | 50.9 |
| 27 | 29 | 28.8 | 28.9 |
| 28 | 24 | 23.8 | 23.9 |
| 29 | 38.6 | 38.2 | 38.4 |
| 30 | 26.8 | 26.4 | 26.6 |
| 31 | 22.4 | 22.4 | 22.4 |
| 32 | 15.9 | 15.7 | 15.8 |
| RNA | |||
|---|---|---|---|
| Standard 1 | 399.52 | ||
| Standard 2 | 9930.43 | ||
| Reading 1 | Reading 2 | Average | |
| 1 | 15.4 | 15.4 | 15.4 |
| 2 | 12.4 | 12.4 | 12.4 |
| 3 | 57.8 | 57.6 | 57.7 |
| 4 | 46.2 | 46.2 | 46.2 |
| 5 | 41.2 | 41.2 | 41.2 |
| 6 | 37.8 | 37.8 | 37.8 |
| 7 | 36.4 | 36.4 | 36.4 |
| 8 | 31.4 | 31.4 | 31.4 |
| 9 | 78 | 77.8 | 77.9 |
| 10 | 48.4 | 48.4 | 48.4 |
| 11 | 54.8 | 54.8 | 54.8 |
| 12 | 48.6 | 48.4 | 48.5 |
| 13 | 23.2 | 23 | 23.1 |
| 14 | 27.4 | 27.4 | 27.4 |
| 15 | 54 | 53.8 | 53.9 |
| 16 | 53 | 52.8 | 52.9 |
| 17 | 16.8 | 16.8 | 16.8 |
| 18 | 15.8 | 15.8 | 15.8 |
| 19 | 41 | 41 | 41 |
| 20 | 37.2 | 37.2 | 37.2 |
| 21 | 13 | 13 | 13 |
| 22 | 13.4 | 13.8 | 13.6 |
| 23 | 13 | 12.8 | 12.9 |
| 24 | 10.8 | 10.6 | 10.7 |
| 25 | 62.6 | 62.6 | 62.6 |
| 26 | 38.4 | 38.2 | 38.3 |
| 27 | 40.2 | 40.2 | 40.2 |
| 28 | 33.6 | 33.6 | 33.6 |
| 29 | 14.8 | 14.8 | 14.8 |
| 30 | 15.2 | 15.2 | 15.2 |
| 31 | 24 | 24 | 24 |
| 32 | 11.8 | 11.8 | 11.8 |
Extractions #1-16:
Extractions #17-32:
TapeStation Results:
| Coral Fragment ID | Extraction ID | Homogenization | RIN |
|---|---|---|---|
| 1754 | 1 | Soft | 8 |
| 1754 | 2 | Hard | 7.7 |
| 1775 | 3 | Soft | 6.8 |
| 1775 | 4 | Hard | 6.9 |
| 2005 | 5 | Soft | 8.2 |
| 2005 | 6 | Hard | 8.1 |
| 2386 | 7 | Soft | 8.2 |
| 2386 | 8 | Hard | 7.8 |
| 2357 | 9 | Soft | 7.8 |
| 2357 | 10 | Hard | 7.3 |
| 1131 | 11 | Soft | 8 |
| 1131 | 12 | Hard | 7.7 |
| 2026 | 13 | Soft | 7.9 |
| 2026 | 14 | Hard | 7.6 |
| 1471 | 15 | Soft | 8.5 |
| 1471 | 16 | Hard | 8 |
| 1644 | 17 | Soft | 8.1 |
| 1644 | 18 | Hard | 8.2 |
| 1676 | 19 | Soft | 7.6 |
| 1676 | 20 | Hard | 7.3 |
| 1631 | 21 | Soft | 8.7 |
| 1631 | 22 | Hard | 8.7 |
| 2183 | 23 | Soft | 8.9 |
| 2183 | 24 | Hard | 8.9 |
| 1103 | 25 | Soft | 8.2 |
| 1103 | 26 | Hard | 7.9 |
| 1696 | 27 | Soft | ** |
| 1696 | 28 | Hard | 8.5 |
| 1600 | 29 | Soft | 9 |
| 1600 | 30 | Hard | 9 |
| 1580 | 31 | Soft | 8.7 |
| 1580 | 32 | Hard | 8.4 |
Link to 20190719 report, Extractions #1-15
Link to 20190719 report, Extractions #16-30
Link to 20190719 report, Extractions #31-32
Link to Agilent 4200 TapeStation System
Gel electrophoresis, qubit, and TapeStation for 16 samples (32 DNA extractions, 32 RNA extractions) takes ~4 hours.