20220303 RNA DNA extractions for unknown coral fragments
DNA/RNA extractions from Putnam Lab collaborator
Extractions of coral fragments
Extraction Date: March 03, 2022
Samples
| Tube number | coll date |
|---|---|
| 2A | unknown |
| 5B | unknown |
| 6A | unknown |
| 54-2 | unknown |
| 54-9 | unknown |
| 69-15 | unknown |
Extraction notes
- Into a bead tube I added 500ul of RNA/DNA shield, a small clipping of fragment, and 300ul of the original sample RNA/DNA shield
- Samples were bead beated for 2 minutes, and then briefly centrifuged to help get rid of some foam.
- Took 300ul from the bead tube and added to a new 1.5ml tube.
- To the 300ul of shield, I added 15ul of ProK, and 30ul of ProK digestion buffer, let sit for 10 minutes
- Spun down samples for 3 minutes at 9000 rcf and then transfer the supernatant to new tube without disturbing the pellet
- 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
- The RNA turned cloudy with a milky white precipitate when I added the 100% ethanol. It seemed to all wash out though
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 | 174.58 | |||
| Standard 2 | 18424.47 | |||
| 2A | nd | nd | nd | |
| 2B | nd | nd | nd | |
| 6A | 3.20 | 3.10 | 3.15 | |
| 54-2 | nd | nd | nd | |
| 54-9 | 6.00 | 5.90 | 5.95 | |
| 69-15 | 11.7 | 11.6 | 11.65 |
RNA
| Tube number | RFU | RNA 1 (ng/uL) | RNA 2 (ng/uL) | Average |
|---|---|---|---|---|
| Standard 1 | 417.33 | |||
| Standard 2 | 10382.34 | |||
| 13 | nd | nd | nd | |
| 15 | nd | nd | nd | |
| 125 | nd | nd | nd | |
| 215 | nd | nd | nd | |
| 243 | nd | nd | nd | |
| 601 | 17.0 | 16.8 | 16.9 |
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
- All terribly degraded
Written on March 3, 2022