Soft and Hard Homogenization Extractions
Soft and Hard Homogenization DNA/RNA Extractions #155-162; #163-178
20190730 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.
Round 1: Extractions #155-162
Fragment preparation start 8:05 end 8:18
Soft/Hard homogenization start 8:19 end 8:43
RNA DNA Extraction start 8:44 end 10:28
Qubit used to check DNA and RNA quantity (ng/μl).
TapeStation used to check RNA quality.
DNA Standard 1: 211.51 ng/μl
DNA Standard 2: 28121.26 ng/μl
RNA Standard 1: 411.35 ng/μl
RNA Standard 2: 11710.89 ng/μl
| Timepoint | Species | Coral ID | Extraction ID | Homogenization | DNA Elution | DNA Reading 1 | DNA Reading 2 | Average DNA ng/μl | RNA Reading 1 | RNA Reading 2 | Average RNA ng/μl | RIN |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 20181103 | Pocillopora | 2873 | 155 | soft | NA | NA | NA | NA | 92.8 | 92.6 | 92.7 | ** |
| 20181103 | Pocillopora | 2873 | 155-1 | soft | 1 | 238 | 238 | 238 | NA | NA | NA | NA |
| 20181103 | Pocillopora | 2873 | 155-2 | soft | 2 | 70.2 | 70 | 70.1 | NA | NA | NA | NA |
| 20181103 | Pocillopora | 2873 | 156 | hard | NA | NA | NA | NA | 55 | 54.4 | 54.7 | 7 |
| 20181103 | Pocillopora | 2873 | 156-1 | hard | 1 | 206 | 206 | 206 | NA | NA | NA | NA |
| 20181103 | Pocillopora | 2873 | 156-2 | hard | 2 | 44.2 | 44.2 | 44.2 | NA | NA | NA | NA |
| 20180923 | Montipora | 1196 | 157 | soft | NA | NA | NA | NA | 33.4 | 33.2 | 33.3 | 9.1 |
| 20180923 | Montipora | 1196 | 157-1 | soft | 1 | 204 | 204 | 204 | NA | NA | NA | NA |
| 20180923 | Montipora | 1196 | 157-2 | soft | 2 | 25.6 | 25.6 | 25.6 | NA | NA | NA | NA |
| 20180923 | Montipora | 1196 | 158 | hard | NA | NA | NA | NA | 20.6 | 20.4 | 20.5 | 8.6 |
| 20180923 | Montipora | 1196 | 158-1 | hard | 1 | 85.2 | 85 | 85.1 | NA | NA | NA | NA |
| 20180923 | Montipora | 1196 | 158-2 | hard | 2 | 17.6 | 17.6 | 17.6 | NA | NA | NA | NA |
| 20181117 | Pocillopora | 1225 | 159 | soft | NA | NA | NA | NA | 106 | 106 | 106 | 7.2 |
| 20181117 | Pocillopora | 1225 | 159-1 | soft | 1 | 171 | 171 | 171 | NA | NA | NA | NA |
| 20181117 | Pocillopora | 1225 | 159-2 | soft | 2 | 43.4 | 43.2 | 43.3 | NA | NA | NA | NA |
| 20181117 | Pocillopora | 1225 | 160 | hard | NA | NA | NA | NA | 56.8 | 56.2 | 56.5 | 6.4 |
| 20181117 | Pocillopora | 1225 | 160-1 | hard | 1 | 112 | 112 | 112 | NA | NA | NA | NA |
| 20181117 | Pocillopora | 1225 | 160-2 | hard | 2 | 21.6 | 21.6 | 21.6 | NA | NA | NA | NA |
| 20181020 | Pocillopora | 2861 | 161 | soft | NA | NA | NA | NA | 82.4 | 82.2 | 82.3 | 7.8 |
| 20181020 | Pocillopora | 2861 | 161-1 | soft | 1 | 274 | 274 | 274 | NA | NA | NA | NA |
| 20181020 | Pocillopora | 2861 | 161-2 | soft | 2 | 34.4 | 34.4 | 34.4 | NA | NA | NA | NA |
| 20181020 | Pocillopora | 2861 | 162 | hard | NA | NA | NA | NA | 56.4 | 56 | 56.2 | 7.7 |
| 20181020 | Pocillopora | 2861 | 162-1 | hard | 1 | 139 | 138 | 138.5 | NA | NA | NA | NA |
| 20181020 | Pocillopora | 2861 | 162-2 | hard | 2 | 25.2 | 25.2 | 25.2 | NA | NA | NA | NA |
Link to 20190730 TapeStation report, Extractions #155-162
Round 2: Extractions #163-178
Fragment preparation start 13:09 end 13:34
Soft/Hard homogenization start 13:35 end 14:05
RNA DNA Extraction start 14:06 end 16:30
Qubit used to check DNA and RNA quantity (ng/μl).
TapeStation used to check RNA quality.
DNA Standard 1: 209.39 ng/μl
DNA Standard 2: 23646.44 ng/μl
RNA Standard 1: 418.45 ng/μl
RNA Standard 2: 11351.13 ng/μl
| Timepoint | Species | Coral ID | Extraction ID | Homogenization | DNA Elution | DNA Reading 1 | DNA Reading 2 | Average DNA ng/μl | RNA Reading 1 | RNA Reading 2 | Average RNA ng/μl | RIN |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 20181103 | Pocillopora | 1302 | 163 | soft | NA | NA | NA | NA | 72.8 | 71.6 | 72.2 | 6.8 |
| 20181103 | Pocillopora | 1302 | 163-1 | soft | 1 | 60.8 | 60.2 | 60.5 | NA | NA | NA | NA |
| 20181103 | Pocillopora | 1302 | 163-2 | soft | 2 | 9.4 | 9.26 | 9.33 | NA | NA | NA | NA |
| 20181103 | Pocillopora | 1302 | 164 | hard | NA | NA | NA | NA | 56 | 55.6 | 55.8 | 7.4 |
| 20181103 | Pocillopora | 1302 | 164-1 | hard | 1 | 51.2 | 49.8 | 50.5 | NA | NA | NA | NA |
| 20181103 | Pocillopora | 1302 | 164-2 | hard | 2 | 10.4 | 10.2 | 10.3 | NA | NA | NA | NA |
| 20181117 | Montipora | 2737 | 165 | soft | NA | NA | NA | NA | 21.4 | 21.2 | 21.3 | 9.2 |
| 20181117 | Montipora | 2737 | 165-1 | soft | 1 | 41.2 | 40.8 | 41 | NA | NA | NA | NA |
| 20181117 | Montipora | 2737 | 165-2 | soft | 2 | 6.64 | 6.54 | 6.59 | NA | NA | NA | NA |
| 20181117 | Montipora | 2737 | 166 | hard | NA | NA | NA | NA | 16.2 | 16 | 16.1 | 9.1 |
| 20181117 | Montipora | 2737 | 166-1 | hard | 1 | 92 | 91.6 | 91.8 | NA | NA | NA | NA |
| 20181117 | Montipora | 2737 | 166-2 | hard | 2 | 6.84 | 6.76 | 6.8 | NA | NA | NA | NA |
| 20181117 | Pocillopora | 2064 | 167 | soft | NA | NA | NA | NA | 69.6 | 69.6 | 69.6 | 8.1 |
| 20181117 | Pocillopora | 2064 | 167-1 | soft | 1 | 284 | 282 | 283 | NA | NA | NA | NA |
| 20181117 | Pocillopora | 2064 | 167-2 | soft | 2 | 29 | 28.8 | 28.9 | NA | NA | NA | NA |
| 20181117 | Pocillopora | 2064 | 168 | hard | NA | NA | NA | NA | 63 | 62.4 | 62.7 | 8 |
| 20181117 | Pocillopora | 2064 | 168-1 | hard | 1 | 208 | 206 | 207 | NA | NA | NA | NA |
| 20181117 | Pocillopora | 2064 | 168-2 | hard | 2 | 28.8 | 30.2 | 29.5 | NA | NA | NA | NA |
| 20181020 | Montipora | 2067 | 169 | soft | NA | NA | NA | NA | ** | ** | ** | ** |
| 20181020 | Montipora | 2067 | 169-1 | soft | 1 | 54 | 53.6 | 53.8 | NA | NA | NA | NA |
| 20181020 | Montipora | 2067 | 169-2 | soft | 2 | 6.76 | 6.7 | 6.73 | NA | NA | NA | NA |
| 20181020 | Montipora | 2067 | 170 | hard | NA | NA | NA | NA | ** | ** | ** | 7.7 |
| 20181020 | Montipora | 2067 | 170-1 | hard | 1 | 44 | 43.8 | 43.9 | NA | NA | NA | NA |
| 20181020 | Montipora | 2067 | 170-2 | hard | 2 | 4.38 | 4.34 | 4.36 | NA | NA | NA | NA |
| 20180923 | Pocillopora | 1168 | 171 | soft | NA | NA | NA | NA | 70.8 | 70 | 70.4 | 8.8 |
| 20180923 | Pocillopora | 1168 | 171-2 | soft | 1 | 232 | 232 | 232 | NA | NA | NA | NA |
| 20180923 | Pocillopora | 1168 | 171-2 | soft | 2 | 16 | 15.9 | 15.95 | NA | NA | NA | NA |
| 20180923 | Pocillopora | 1168 | 172 | hard | NA | NA | NA | NA | 60.4 | 59.8 | 60.1 | 8.8 |
| 20180923 | Pocillopora | 1168 | 172-1 | hard | 1 | 125 | 125 | 125 | NA | NA | NA | NA |
| 20180923 | Pocillopora | 1168 | 172-2 | hard | 2 | 21.6 | 21.4 | 21.5 | NA | NA | NA | NA |
| 20181117 | Montipora | 1561 | 173 | soft | NA | NA | NA | NA | 31.2 | 30.2 | 30.7 | 8.6 |
| 20181117 | Montipora | 1561 | 173-1 | soft | 1 | 398 | 396 | 397 | NA | NA | NA | NA |
| 20181117 | Montipora | 1561 | 173-2 | soft | 2 | 22.2 | 22.2 | 22.2 | NA | NA | NA | NA |
| 20181117 | Montipora | 1561 | 174 | hard | NA | NA | NA | NA | 17.6 | 17.4 | 17.5 | 8.8 |
| 20181117 | Montipora | 1561 | 174-1 | hard | 1 | 64.2 | 63.8 | 64 | NA | NA | NA | NA |
| 20181117 | Montipora | 1561 | 174-2 | hard | 2 | 16 | 15.9 | 15.95 | NA | NA | NA | NA |
| 20180922 | Pocillopora | 1220 | 175 | soft | NA | NA | NA | NA | 79.6 | 78.8 | 79.2 | 8.8 |
| 20180922 | Pocillopora | 1220 | 175-1 | soft | 1 | 86.2 | 85.8 | 86 | NA | NA | NA | NA |
| 20180922 | Pocillopora | 1220 | 175-2 | soft | 2 | 24.4 | 24.2 | 24.3 | NA | NA | NA | NA |
| 20180922 | Pocillopora | 1220 | 176 | hard | NA | NA | NA | NA | 57.2 | 56.8 | 57 | 7.8 |
| 20180922 | Pocillopora | 1220 | 176-1 | hard | 1 | 109 | 109 | 109 | NA | NA | NA | NA |
| 20180922 | Pocillopora | 1220 | 176-2 | hard | 2 | 20.4 | 20.2 | 20.3 | NA | NA | NA | NA |
| 20180922 | Montipora | 2000 | 177 | soft | NA | NA | NA | NA | 28.4 | 28 | 28.2 | ** |
| 20180922 | Montipora | 2000 | 177-1 | soft | 1 | 124 | 122 | 123 | NA | NA | NA | NA |
| 20180922 | Montipora | 2000 | 177-2 | soft | 2 | 12.2 | 12.1 | 12.15 | NA | NA | NA | NA |
| 20180922 | Montipora | 2000 | 178 | hard | NA | NA | NA | NA | 19.4 | 19 | 19.2 | 7.4 |
| 20180922 | Montipora | 2000 | 178-1 | hard | 1 | 128 | 128 | 128 | NA | NA | NA | NA |
| 20180922 | Montipora | 2000 | 178-2 | hard | 2 | 19.2 | 18.9 | 19.05 | NA | NA | NA | NA |
Link to 20190730 TapeStation report, Extractions #163-178
Written on July 30, 2019