Thursday, April 2, 2015

4 2 2015 EZNA Seed Isolation with Fidalgo pt 2.

Today I completed the 96 seed isolation with the EZNA kit. I isolated the last 12 samples for Fidalgo. The samples are in the freezer in 209 and are ready for GBS processing. You can read about the last Fidalgo isolation in this blogpost.

Before using the EZNA Kit I dissected out whole body tissue from 12 seed oysters into the homogenization tube. The oysters are labeled NF 21-32. I used flame sterilized equipment to dissect the animals. 

The protocol is as follows:

  1. Added 350 ul ML1 Buffer
  2. Added 25 ul Proteinase K solution
  3. Used pestle in homogenization tube to grind tissue in solution
  4. Vortexed
  5. Incubated at 60 C for 30 minutes
  6. Added 350 ul Phenol:Chloroform:Isoamyl Alcohol (25:24:1)
  7. Vortexed
  8. Centrifuged 10,000 g for 2 minutes
  9. Transferred the upper aqueous phase to new tube (~300 ul)
  10. Added 300 ul MBL Buffer
  11. Added 10 ul RNase A
  12. Vortexed for 15 seconds
  13. Incubated at 70C (started at 67.5 C) for 10 minutes
  14. Cooled to room temperature sitting for 5 minutes
  15. Added 300 ul 100% EtOH
  16. Vortexed for 15 seconds
  17. Put spin column in collection tube
  18. Added 750 ul sample solution to column
  19. Centrifuged at 10,000 g for 1 minute at 4C
  20. Discarded flowthrough
  21. Repeated 18-20 with remaining sample
  22. Discarded collection tube and replaced with a new one. 
  23. Added 500 ul HBC solution. 
  24. Centrifuged at 10,000 g for 30 seconds at 4C
  25. Discarded flowthrough
  26. Added 700 ul DNA Wash Buffer
  27. Centrifuged at 10,000 g for 1 minute at 4C
  28. Discarded flowthrough
  29. Repeated 26-28
  30. Centrifuged Empty column for 2 minutes at 10,000 g at 4C
  31. Discarded collection tube and put column into microcentrifuge tube for sample collection
  32. Added 100 ul preheated 70C Elution Buffer
  33. Incubated for 2 minutes
  34. Centrifuged at 10,000 g for 1 minute at 4C
  35. Repeated 32-34. 
  36. Stored DNA at -20 C

DNA is in a box labelled:
Seed Oly DNA HL NF
3/23/15 Jake Heare 1 of 2

Seed Oly DNA SN 
3/31/15 JH 2 of 2

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