| layout | title | date | categories | tags |
|---|---|---|---|---|
post |
20211008 RNA DNA extractions from E5 project |
2021-10-08 |
Processing |
DNA RNA |
Extractions from the three coral species from each of the four timepoints
Samples
| Tube number | Timepoint | Species | Colony ID | Coll date | Site |
|---|---|---|---|---|---|
| 23 | January | Porites | POR-236 | 20200103 | 2 |
| 39 | January | Acropora | ACR-244 | 20200103 | 2 |
| 73 | January | Pocillopora | POC-200 | 20200103 | 2 |
| 421 | March | Porites | POR-82 | 20200303 | 1 |
| 433 | March | Acropora | ACR-343 | 20200304 | 3 |
| 457 | March | Pocillopora | POC-378 | 20200304 | 3 |
| 523 | Sept | Porites | POR-251 | 20200910 | 2 |
| 607 | Sept | Porites | POR-383 | 20200908 | 3 |
| 615 | Sept | Porites | POR-391 | 20200908 | 3 |
| 839 | November | Porites | POR-341 | 20201031 | 3 |
| 847 | November | Pocillopora | POC-377 | 20201031 | 3 |
| 853 | November | Acropora | ACR-343 | 20201031 | 3 |
Tube 73 had a cap that said 73, but the side of the tube said 74
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 | 197.80 | |||
| Standard 2 | 23245.31 | |||
| 23 | nd | nd | nd | |
| 39 | 31.4 | 30.8 | 31.1 | |
| 73 | 59.4 | 59.0 | 59.2 | |
| 421 | 3.46 | 3.36 | 3.41 | |
| 433 | 24.6 | 23.8 | 24.2 | |
| 457 | 33.6 | 33.4 | 33.5 | |
| 523 | 4.10 | 4.00 | 4.05 | |
| 607 | 26.4 | 26.6 | 26.5 | |
| 615 | 13.5 | 13.6 | 13.55 | |
| 839 | 6.02 | 5.82 | 5.92 | |
| 847 | 2.48 | 2.36 | 2.42 | |
| 853 | 45.2 | 44.6 | 44.9 |
RNA
| Tube number | RFU | RNA 1 (ng/uL) | RNA 2 (ng/uL) | Average |
|---|---|---|---|---|
| Standard 1 | 372.52 | |||
| Standard 2 | 6731.91 | |||
| 23 | 13.4 | 13.2 | 13.3 | |
| 39 | 14.0 | 13.4 | 13.7 | |
| 73 | 42.6 | 42.4 | 42.5 | |
| 421 | 23.6 | 24.2 | 23.9 | |
| 433 | 14.4 | 13.6 | 14.0 | |
| 457 | 29.6 | 30.4 | 30.0 | |
| 523 | 14.6 | 14.8 | 14.7 | |
| 607 | 21.2 | 21.0 | 21.1 | |
| 615 | 37.6 | 37.6 | 37.6 | |
| 839 | 15.0 | 14.8 | 14.9 | |
| 847 | 49.0 | 48.8 | 48.9 | |
| 853 | 25.0 | 24.8 | 24.9 |
Tape Station
- Used to check RNA quality Protocol
- Results Link
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
- NA