20220211 RNA DNA extractions from E5 project
DNA/RNA extractions from E5 project
Re-Extractions from Acropora from three of the timepoints, plus 4 samples from Maggie?
Extraction Date: February 11, 2022
Samples
| Tube number | Timepoint | Species | Colony ID | Coll date | Site |
|---|---|---|---|---|---|
| 141 | January | Acropora | ACR-266 | 20200106 | 3 |
| 153 | January | Acropora | ACR-209 | 20200106 | 3 |
| 291 | March | Acropora | ACR-381 | 20200303 | 2 |
| 467 | March | Acropora | ACR-76 | 20200305 | 1 |
| 717 | November | Acropora | ACR-235 | 20201101 | 1 |
| 775 | November | Acropora | ACR-75 | 20201101 | 1 |
| 817 | November | Acropora | ACR-253 | 20201031 | 3 |
| 835 | November | Acropora | ACR-216 | 20201031 | 3 |
| 2A | NA | unk | NA | NA | NA |
| 5B | NA | unk | NA | NA | NA |
| 6A | NA | unk | NA | NA | NA |
| 54-9 | NA | unk | NA | NA | NA |
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 | 197.77 | |||
| Standard 2 | 21529.84 | |||
| 141 | 5.80 | 5.78 | 5.79 | |
| 153 | 3.04 | 3.24 | 3.14 | |
| 291 | 63.8 | 62.8 | 63.3 | |
| 467 | 17.4 | 17.4 | 17.4 | |
| 717 | 18.4 | 18.0 | 18.2 | |
| 775 | 8.88 | 8.68 | 8.78 | |
| 817 | 11.6 | 11.4 | 11.5 | |
| 835 | 18.3 | 18.5 | 18.4 | |
| 2A | nd | nd | nd | |
| 5B | nd | nd | nd | |
| 6A | nd | nd | nd | |
| 54-9 | nd | nd | nd |
RNA
| Tube number | RFU | RNA 1 (ng/uL) | RNA 2 (ng/uL) | Average |
|---|---|---|---|---|
| Standard 1 | 421.60 | |||
| Standard 2 | 10814.86 | |||
| 141 | nd | nd | nd | |
| 153 | nd | nd | nd | |
| 291 | 10.4 | 10.4 | 10.4 | |
| 467 | 12.2 | 11.8 | 12.0 | |
| 717 | nd | nd | nd | |
| 775 | nd | nd | nd | |
| 817 | 12.2 | 12.6 | 12.4 | |
| 835 | nd | nd | nd | |
| 2A | nd | nd | nd | |
| 5B | nd | nd | nd | |
| 6A | nd | nd | nd | |
| 54-9 | 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
Written on February 11, 2022