| layout | title | date | categories | tags |
|---|---|---|---|---|
post |
20220201 RNA DNA extractions from E5 project |
2022-02-01 |
Processing |
DNA RNA |
Re-Extractions from Porites from each of the four timepoints
Samples
| Tube number | Timepoint | Species | Colony ID | Coll date | Site |
|---|---|---|---|---|---|
| 1 | January | Porites | POR-266 | 20200103 | 2 |
| 25 | January | Porites | POR-209 | 20200103 | 2 |
| 111 | January | Porites | POR-381 | 20200106 | 3 |
| 201 | January | Porites | POR-76 | 20200110 | 1 |
| 287 | March | Porites | POR-235 | 20200303 | 2 |
| 415 | March | Porites | POR-75 | 20200305 | 1 |
| 497 | Sept | Porites | POR-253 | 20200910 | 2 |
| 515 | Sept | Porites | POR-216 | 20200910 | 2 |
| 633 | Sept | Porites | POR-73 | 20200911 | 1 |
| 693 | Sept | Porites | POR-80 | 20200911 | 1 |
| 739 | November | Porites | POR-70 | 20201101 | 1 |
| 907 | November | Porites | POR-221 | 20201030 | 2 |
Extraction notes
- I looked at each sample, for those samples that were darker (287, 415, 515, 633, 693) I took 100ul of sample and added it to 200ul of new shield
- For samples that were lighter in pigment, (1, 25, 111, 201, 739, 907) I took 200ul of sample and added to 100ul 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 | 201.28 | |||
| Standard 2 | 23737.21 | |||
| 1 | 4.30 | 4.10 | 4.20 | |
| 25 | 2.60 | 2.44 | 2.52 | |
| 111 | 3.08 | 2.90 | 2.99 | |
| 201 | 2.10 | 2.00 | 2.05 | |
| 287 | 2.08 | nd | 2.00 | |
| 415 | 2.36 | 2.20 | 2.28 | |
| 497 | 12.8 | 12.4 | 12.6 | |
| 515 | 2.04 | nd | 2.00 | |
| 633 | 3.72 | 3.52 | 3.62 | |
| 693 | 2.54 | 2.42 | 2.48 | |
| 739 | 2.10 | nd | 2.00 | |
| 907 | 8.92 | 8.74 | 8.82 |
RNA
| Tube number | RFU | RNA 1 (ng/uL) | RNA 2 (ng/uL) | Average |
|---|---|---|---|---|
| Standard 1 | 423.11 | |||
| Standard 2 | 10529.05 | |||
| 1 | nd | nd | nd | |
| 25 | nd | nd | nd | |
| 111 | nd | nd | nd | |
| 201 | 19.4 | 19.8 | 19.6 | |
| 287 | 13.8 | 14.6 | 14.2 | |
| 415 | 13.0 | 13.0 | 13.0 | |
| 497 | nd | nd | nd | |
| 515 | 14.8 | 14.6 | 14.7 | |
| 633 | 18.4 | 18.2 | 18.3 | |
| 693 | 16.8 | 16.6 | 16.7 | |
| 739 | 22.4 | 22.2 | 22.3 | |
| 907 | nd | nd | nd |
Tape Station
- Tape station broken!
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