Soft and Hard Homogenization Extractions
Soft and Hard Homogenization DNA/RNA Extractions #255-266
20190802 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.
DNA samples atypically put in green spin-away columns (green columns are usually used for RNA samples), but Zymo webpage states green spin-away columns can be used for DNA and/or RNA. DNA samples measured on the Qubit before proceeding with RNA samples. DNA/RNA shield atypically used instead of Wash Buffer during DNA extraction.
DNA samples #260 and #266 have final elution volume of 80 μl.
Fragment preparation start 9:25 end 9:43
Soft/Hard homogenization start 9:44 end 10:10
RNA DNA Extraction start 10:11 end 12:51
Qubit used to check DNA and RNA quantity (ng/μl).
TapeStation used to check RNA quality.
DNA Standard 1: 188.62 ng/μl
DNA Standard 2: 18,548.07 ng/μl
RNA Standard 1: 389.8 ng/μl
RNA Standard 2: 10,185.69 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 |
|---|---|---|---|---|---|---|---|---|---|---|---|
| 20180929 | Pocillopora | 1468 | 255 | soft | 6.32 | 6.16 | 6.24 | 131 | 130 | 130.5 | 6.7 |
| 20180929 | Pocillopora | 1468 | 256 | hard | 10.4 | 10.3 | 10.35 | 78 | 77.8 | 77.9 | 6.5 |
| 20181020 | Pocillopora | 1709 | 257 | soft | 8.48 | 8.46 | 8.47 | 77.4 | 77.2 | 77.3 | 7.7 |
| 20181020 | Pocillopora | 1709 | 258 | hard | 6.82 | 6.78 | 6.8 | 70.2 | 70 | 70.1 | 7 |
| 20180922 | Montipora | 1277 | 259 | soft | 7.68 | 7.62 | 7.65 | 48.6 | 48.6 | 48.6 | 8.9 |
| 20180922 | Montipora | 1277 | 260 | hard | 5.96 | 5.94 | 5.95 | 27.6 | 27.6 | 27.6 | 8.9 |
| 20181020 | Pocillopora | 1329 | 261 | soft | 4.96 | 4.94 | 4.95 | 74.6 | 74.4 | 74.5 | 8.3 |
| 20181020 | Pocillopora | 1329 | 262 | hard | 6.38 | 6.36 | 6.37 | 71 | 71 | 71 | 7.7 |
| 20180922 | Montipora | 1128 | 263 | soft | 10.7 | 10.6 | 10.65 | 47 | 47 | 47 | 8.7 |
| 20180922 | Montipora | 1128 | 264 | hard | 3.74 | 3.74 | 3.74 | 30.2 | 30.4 | 30.3 | 8.9 |
| 20180929 | Montipora | 1588 | 265 | soft | 8.96 | 8.94 | 8.95 | 25.2 | 25.2 | 25.2 | 9.4 |
| 20180929 | Montipora | 1588 | 266 | hard | 4.66 | 4.66 | 4.66 | 20 | 20 | 20 | 9.6 |
Link to 20190802 TapeStation report, Extractions #255-266
