DNA Only Extraction of 16 Sea Star Samples
DNA Extractions of 16 Hystera and Pentagona With the Zymo Research Quick-DNA Miniprep Plus Kit
Notes: followed kit recommendations for samples stored in DNA/RNA Shield and used and intermediate incubation time of 2 hours
Sample Preparation and Digestion
- Samples:
- COPI-010
- CPOI-009
- CPOI-005
- CPOI-001
- CPBP-007
- CPBP-005
- CPBP-004
- CPBO-008
- CPBO-004
- CPAB-010
- CPAB-006
- CPAB-002
- CPDB-007
- CPDB-004
- CHSY-008
- CHSY-009
- Thawed samples on ice bucket
- Prepared forceps, foil, and razor blades
- Cleaned with 10% bleach, DI water, and 70% ethanol before and between each sample
- Prepared a 1.5mL tube for each sample with 300ul DNA/RNA shield in each
- Cut a small piece of tissue for each sample then minced with the razor blade until it was flat and could come apart a little bit and placed in their respective 1.5mL tube note sample CPBO 008 did not have tissue, it looked like it had broken up completely into the liquid so I added 200ul of the liquid to the extraction tube
- Added 150ul of the blue solid tissue buffer to each sample processing tube
- Added 10ul of proteinase K enzyme to each sample processing tube
- Vortexed and spun down tubes
- Placed tubes in the thermomixer at 55 degrees C for 2 hours shaking at 1200rpm
- Placed all sample tubes in the tabletop centrifuge and spun at 16,000rcf for 1 minute
- Removed ~450ul of supernatant into new 1.5mL tubes note, sample CPBO 008 looks like it lost liquid during incubation because less than 400ul were present
- Added 450ul of Genomic DNA Binding Buffer to each new tube
- Vortexed and spun down tubes
DNA Extraction
- Set up 1 spin column and collection tube per sample
- Warmed 10mM Tris HCl to 70 degrees C in the thermomixer
- Added 700ul of each sample to their labeled spin columns
- Centrifuged at 16000 rcf for 1 min
- Discarded flow through
- Added the rest of the liquid to the spin columns and centrifuged in the same way
- Transferred columns to new collection tubes
- Added 400ul DNA pre-wash buffer to each column
- Centrifuged 16000 rcf for 1 minute and discarded flow through
- Added 700ul g-DNA wash buffer to each column
- Centrifuged 16000 rcf for 1 minute and discarded flow through
- Added 200ul g-DNA wash buffer to each column
- Centrifuged 16000 rcf for 1 minute and discarded flow through
- Made 1.5mL tubes labeled completely with sample names and information
- Transferred spin columns to those 1.5mL tubes
- Added 50ul of warmed 10mM tris HCl by dripping to each spin column filter
- Incubated columns for 5 minutes
- Centrifuged columns for 1 min at 16000 rcf
- Repeated last 3 steps once
- Placed tubes on ice afterwards
Qubit
- Broad range dsDNA qubit (protocol)
- Amounts are in ng/ul and samples were read twice
| Sample | Reading 1 | Reading 2 | Average DNA (ng/ul) |
|---|---|---|---|
| standard 1 | 183 | - | - |
| standard 2 | 20040 | - | - |
| CPOI-010 | 45.4 | 44.8 | 45.1 |
| CPOI-009 | 34.8 | 34.8 | 34.8 |
| CPOI-005 | 40.2 | 40 | 40.1 |
| CPOI-001 | 26.8 | 26.6 | 26.7 |
| CPBP-007 | 37.2 | 36.6 | 36.9 |
| CPBP-005 | 40 | 39.8 | 39.9 |
| CPBP-004 | 56.4 | 55.8 | 56.1 |
| CPBO-008 | too low | - | - |
| CPBO-004 | 7.8 | 7.8 | 7.8 |
| CPAB-010 | 26.8 | 26.6 | 26.7 |
| CPAB-006 | 47.8 | 47.4 | 47.6 |
| CPAB-002 | 33 | 32.8 | 32.9 |
| CPDB-007 | 39.8 | 39.4 | 39.6 |
| CPDB-004 | 34.8 | 36.4 | 35.6 |
| CHSY-008 | 33 | 33 | 33 |
| CPSY-009 | 13.5 | 13.5 | 13.5 |
Gel
Written on December 11, 2020