Monday, May 12, 2014

DNA Extraction Attempt 2 5-12-14

Attempting to extract DNA from 5-10 mm whole oysters minus the shell. This is needed so that sequencing can occur in the future with completed extractions. Unlike the last attempt I will be switching out 95% EtOH for Isopropanol to get better extraction results.

Protocol is as follows:

Incubation for 24 hours at room temp:

2.35ulProteinase K (100ug/ml)
1wholeoyster 5-10 mm
  1. Pipette 1 ul DNAzol and 2.35 ul Proteinase K in tube containing oyster
  2. Homogenize each oyster with disposable pestle.
  3. Centrifuge briefly
  4. Incubate overnight at ROOM TEMP.


1ml75% EtOH
1ml75% EtOH
200ulSterile H2O

  1. Centrifuge @ 10,000 g for 10 minutes.
  2. Collect and Transfer Supernant; Discard pellet
  3. Add 500 ul Isopropanol
  4. Mix through inversion
  5. Incubate for 1 minute at ROOM TEMP.
  6. Spin down DNA precipitate
  7. Remove supernant
  8. Wash with 1 ml 75% EtOH
  9. Spin down
  10. Remove supernant
  11. Wash with 1 ml 75% EtOH
  12. Spin down until solid pellet
  13. Elute with 200 ul Sterile H2O
  14. Quant.

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