2nd Try Qiagen Genomic Tip HMW DNA Extraction of Fire Coral
2nd Attempt of HMW DNA Extraction of Fire Coral Stored in DNA/RNA Shield
Goal: Extract high quality DNA from these samples, and try to get 20-25ug of material
Result: ~3ug of DNA, not enough, quality is pretty good
Thoughts: Not sure how much I will be able to get out of these samples, I have enough for one more extraction. I thought using the DNA/RNA shield from the sample tubes would have helped a lot, but it seems to have only helped minimally. There could be some incompatibilities with the buffer and the shield.
Extraction and Processing
- Followed HMW DNA protocol almost exactly, notes following
- 0.4g of “chunk” or coral was used:
- Sample once ground:
- I was thinking that maybe part of the reason that the yield was so low the first time was because these samples were stored in DNA/RNA shield, which is partly a lysis buffer. And so I thought that it was possible that a lot of the tissue had been “lysed” off the skeleton and was in solution in the liquid. There was what looked sort of like lysed tissue in the bottom of the sample tubes (see below picture), so I also added 800ul of the liquid from the sample tube to the extraction (the tube with the crushed sample and the buffer G2 mix)
- Sample tube with “residue” at the bottom:
- All other steps performed as the protocol states
- There was a very small pellet visible after the ethanol wash in the precipitation. Tube 10 had very little DNA, I likely sucked up some of the pellet on that one
QC 20210420
Qubit
- Broad Range Qubit the next day (protocol)
- Flicked tube and took from both top T and bottom B of the tube measurements
| Standard 1 | Standard 2 | Sample | Average DNA ng/µl | Averaged top and bottom ng/ul |
|---|---|---|---|---|
| 173 RFU | 19063 RFU | 7 T | 11.6 | |
| - | - | 7 B | 12.25 | 11.925 |
| - | - | 8 T | 12.45 | |
| - | - | 8 B | 13.8 | 13.125 |
| - | - | 9 T | 12.1 | |
| - | - | 9 B | 13.05 | 12.575 |
| - | - | 10 T | 2.31 | |
| - | - | 10 B | 2.5 | 2.405 |
| - | - | 11 T | 12.3 | |
| - | - | 11 B | 13 | 12.65 |
| - | - | 12 T | 13.65 | |
| - | - | 12 B | 13.4 | 13.525 |
Genomic DNA TapeStation
- Followed Tapestation protocol
- Results link
- Quality is pretty good, they’re not at the above 60,000bp mark but there isn’t a lot of low smearing either
Total Amount of DNA from this extraction based on average top and bottom Qubit values and ~47ul of sample volume in each tube
- 7: 560.475ng DNA
- 8: 616.875ng DNA
- 9: 591.025ng DNA
- 10: 113.035ng DNA
- 11: 594.55ng DNA
- 12: 635.675ng DNA
- TOTAL: 3111.635ng DNA, 3.111ug DNA
Written on April 20, 2021