DNA Only Extraction of Last 8 Sea Star Samples
The Last Set of DNA Extractions with 8 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:
- CHSB-005
- CHSB-007
- CHSB-009
- COAB2-002
- CPAB-009
- CPBO-007
- CPBO-008
- CPBP-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
- Samples CHSB-005 and CPBO-008 only ha liquid in the tubes, the sample had broken up into the DNA/RNA Shield, so 300ul of that liquid was used instead of new shield
- 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
- 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 | 185 | - | - |
| standard 2 | 18957 | - | - |
| CHSB-005 | 4.66 | 4.58 | 4.62 |
| CHSB-007 | 21 | 21 | 21 |
| CHSB-009 | 20.2 | 20.2 | 20.2 |
| CPAB2-002 | 29.8 | 29.6 | 29.7 |
| CPAB-009 | 26.4 | 26.2 | 26.3 |
| CPBO-007 | 23 | 22.8 | 22.9 |
| CPBO-008 | 12.4 | 12.4 | 12.4 |
| CPBP-009 | 46.2 | 46 | 46.1 |
Gel
Written on December 16, 2020