20220221 RNA DNA extractions from E5 project
DNA/RNA extractions from E5 project
Re-Extractions from Acropora and Pocillopora from three of the timepoints
Extraction Date: February 21, 2022
Samples
| Tube number | Timepoint | Species | Colony ID | Coll date | Site |
|---|---|---|---|---|---|
| 85 | January | Acropora | ACR-351 | 20200106 | 3 |
| 207 | January | Acropora | ACR-140 | 20200110 | 1 |
| 219 | January | Acropora | ACR-165 | 20200110 | 1 |
| 227 | January | Acropora | ACR-190 | 20200305 | 1 |
| 229 | January | Acropora | ACR-175 | 20200110 | 1 |
| 249 | January | Acropora | ACR-186 | 20200110 | 1 |
| 263 | January | Acropora | ACR-187 | 20200110 | 1 |
| 343 | March | Acropora | ACR-360 | 20200304 | 3 |
| 347 | March | Acropora | ACR-350 | 20200304 | 3 |
| 213 | January | Pocillopora | POC-52 | 20200110 | 1 |
| 255 | January | Pocillopora | POC-55 | 20200110 | 1 |
| 537 | Sept | Pocillopora | POC-255 | 20200910 | 2 |
Extraction notes
- For each sample I took 200ul the RNA/DNA shield and added it to a new 1.5ml tube containing 100ul of new RNA/DNA 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 | 152.95 | |||
| Standard 2 | 15716.62 | |||
| 85 | 12.8 | 12.4 | 12.6 | |
| 207 | 13.0 | 12.7 | 12.85 | |
| 219 | 10.9 | 10.6 | 10.75 | |
| 227 | 10.0 | 9.66 | 9.83 | |
| 229 | 4.20 | 3.98 | 4.09 | |
| 249 | 11.2 | 10.9 | 11.05 | |
| 263 | 18.1 | 17.9 | 18.0 | |
| 343 | 4.10 | 3.98 | 4.04 | |
| 347 | 18.7 | 18.3 | 18.5 | |
| 213 | 4.46 | 4.28 | 4.37 | |
| 255 | 4.14 | 4.02 | 4.08 | |
| 537 | 5.50 | 5.34 | 5.42 |
RNA
| Tube number | RFU | RNA 1 (ng/uL) | RNA 2 (ng/uL) | Average |
|---|---|---|---|---|
| Standard 1 | 426.61 | |||
| Standard 2 | 10583.93 | |||
| 85 | nd | nd | nd | |
| 207 | nd | nd | nd | |
| 219 | nd | nd | nd | |
| 227 | nd | nd | nd | |
| 229 | nd | nd | nd | |
| 249 | nd | nd | nd | |
| 263 | nd | nd | nd | |
| 343 | nd | nd | nd | |
| 347 | nd | nd | nd | |
| 213 | nd | nd | nd | |
| 255 | nd | nd | nd | |
| 537 | 26.2 | 26.4 | 26.3 |
Tape Station
- Not run
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
- The RNA looks really degraded on the gel. I wouldn’t trust the reading of 537. 213 looks the best out of all of them.
Written on February 21, 2022