Before using the EZNA Kit I dissected out whole body tissue from 20 seed oysters into the homogenization tube. The oysters are labeled HL1-20. I used flame sterilized equipment to dissect the animals.
The protocol is as follows:
- Added 350 ul ML1 Buffer
- Added 25 ul Proteinase K solution
- Used pestle in homogenization tube to grind tissue in solution
- Vortexed
- Incubated at 60 C for 30 minutes
- Added 350 ul Phenol:Chloroform:Isoamyl Alcohol (25:24:1)
- Vortexed
- Centrifuged 10,000 g for 2 minutes
- Transferred the upper aqueous phase to new tube (~300 ul)
- Added 300 ul MBL Buffer
- Added 10 ul RNase A
- Vortexed for 15 seconds
- Incubated at 70C (started at 67.5 C) for 10 minutes
- Cooled to room temperature sitting for 5 minutes
- Added 300 ul 100% EtOH
- Vortexed for 15 seconds
- Put spin column in collection tube
- Added 750 ul sample solution to column
- Centrifuged at 10,000 g for 1 minute at 4C
- Discarded flowthrough
- Repeated 18-20 with remaining sample
- Discarded collection tube and replaced with a new one.
- Added 500 ul HBC solution.
- Centrifuged at 10,000 g for 30 seconds at 4C
- Discarded flowthrough
- Added 700 ul DNA Wash Buffer
- Centrifuged at 10,000 g for 1 minute at 4C
- Discarded flowthrough
- Repeated 26-28
- Centrifuged Empty column for 2 minutes at 10,000 g at 4C
- Discarded collection tube and put column into microcentrifuge tube for sample collection
- Added 100 ul preheated 70C Elution Buffer
- Incubated for 2 minutes
- Centrifuged at 10,000 g for 1 minute at 4C
- Repeated 32-34.
- Stored DNA at -20 C
Tomorrow I will run the DNA out on a gel to check for quality.
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