20211130 RNA DNA extractions from E5 project
DNA/RNA extractions from E5 project
Extractions from the three coral species from the first timepoints
Extraction Date: November 30, 2021
Samples
| Tube number | Timepoint | Species | Colony ID | Coll date | Site |
|---|---|---|---|---|---|
| 195 | January | Porites | POC-346 | 20200110 | 1 |
| 199 | January | Pocillopora | ACR-396 | 20200110 | 1 |
| 203 | January | Acropora | POR-383 | 20200110 | 1 |
| 205 | January | Pocillopora | POC-257 | 20200110 | 1 |
| 207 | January | Acropora | POR-253 | 20200110 | 1 |
| 209 | January | Porites | POR-266 | 20200110 | 1 |
| 211 | January | Acropora | POC-371 | 20200110 | 1 |
| 215 | January | Porites | POR-362 | 20200110 | 1 |
| 225 | January | Acropora | ACR-210 | 20200110 | 1 |
| 227 | January | Acropora | POC-55 | 20200110 | 1 |
| 229 | January | Acropora | POR-240 | 20200110 | 1 |
| 10S | N/A | Acropora | 10S | N/A | N/A |
Extraction notes
- ACR and POC samples: pulled out 300ul of shield
- POR samples: pulled out 150ul of shield and added to 150ul of new shield
- Spun down samples for 3 minutes at 9000 rcf and then transfer the supernatant to new tube without disturbing the pellet
- 300ul of shield, 15ul of ProK, and 30ul of ProK digestion buffer, let sit for 2 minutes
- All spins were done for 1 minute or 2.30 minutes
- Did two washes with 700ul of wash buffer for both the DNA and RNA
- Then followed the protocol as described in protocol
Qubit
- Used Broad range dsDNA and RNA Qubit Protocol
- All samples read twice, standard only read once
DNA
| Tube number | RFU | DNA 1 (ng/uL) | DNA 2 (ng/uL) | Average |
|---|---|---|---|---|
| Standard 1 | 195.83 | |||
| Standard 2 | 28772.93 | |||
| 195 | nd | nd | nd | |
| 199 | 13.0 | 13.0 | 13.0 | |
| 203 | 25.0 | 25.0 | 25.0 | |
| 205 | 21.0 | 20.8 | 20.9 | |
| 207 | 16.5 | 16.6 | 16.65 | |
| 209 | nd | nd | nd | |
| 211 | 11.4 | 11.3 | 11.35 | |
| 215 | nd | nd | nd | |
| 225 | 10.5 | 10.8 | 10.65 | |
| 227 | 12.7 | 12.7 | 12.7 | |
| 229 | 19.5 | 19.4 | 19.45 | |
| 10S | 14.0 | 14.0 | 14.0 |
RNA
| Tube number | RFU | RNA 1 (ng/uL) | RNA 2 (ng/uL) | Average |
|---|---|---|---|---|
| Standard 1 | 409.39 | |||
| Standard 2 | 13867.92 | |||
| 195 | 11.0 | 11.4 | 11.2 | |
| 199 | 11.0 | 11.0 | 11.0 | |
| 203 | nd | nd | nd | |
| 205 | 14.2 | 14.2 | 14.2 | |
| 207 | nd | nd | nd | |
| 209 | 15.6 | 15.6 | 15.6 | |
| 211 | nd | nd | nd | |
| 215 | nd | nd | nd | |
| 225 | nd | nd | nd | |
| 227 | nd | nd | nd | |
| 229 | nd | nd | nd | |
| 10S | nd | nd | nd |
Tape Station
- Tape station broke!
Gel
- Modified from this protocol
- Added 0.75g of agarose and 50ml of 1x TAE to flask and microwaved for 45 seconds. This makes a 1.5% gel
- Once cool enough to touch added 2ul of gel green stain
- Swirled and poured into gel mould with comb
- Once solidified, covered with 1X TAE as a running buffer
- Added 1ul of purple loading dye to each of my QC strip tube samples. I had ~9ul of DNA leftover from QC and ~8ul of RNA
- Loaded my gel with the DNA first, then skipped a well and then the RNA
- Ran the gel for 60 minutes at 60 volts
Addtional Notes
- N/A
Written on November 30, 2021