Before the summer of 2018, we used Qiagen DNeasy 96 well extraction kits

Samples were lysed and DNA extracted following the protocol in Ali et al. 2016.

Special notes:

The 26% salt solution is 4.45 Molar.

Supplies needed

Extraction Methods

2019-09-05

Prepare a solution of Lifton’s Buffer

Prepare Lifton’s buffer with proteinase K

Prepare hybridization solution

Prepare low TE solution

Turn on the incubator ## 2018-07-26 Load samples for lysis
1. Add 80uL Lifton’s buffer to each well of a 96 well plate.
2. Add fin clips measuring 2-25mm^2 to each well.
3. Add 40uL enzyme solution to each well, pipet up and down to mix.
4. Seal plate and incubate at 55C overnight.

2018-07-27

Extract DNA from lysate
1. To a new plate add 45uL hybridization buffer to each well.
2. Add 15uL Agencourt Ampure beads.
3. Add 45uL of the lysate and pipet up and down to mix.
4. Incubate at room temperature for 5 minutes, then place on magnet and allow the wells to clear.
5. Remove supernatant.
6. Remove from the magnet and use 150uL freshly prepared 80% EtOH to resuspend the beads.
7. Wash with ethanol again
8. wash with ethanol a 3rd time.
9. Allow beads to air dry on the magnet
10. Add 45uL low TE to elute DNA from the beads

Organize samples into plate format:

  1. Sample_id plate locations
  1. Extraction_id plate locations

Write the newly assigned extraction_ids to the database

Check out a generic DNA cleaner recipe from Bridgett vonHoldt to replace the very expensive version we use here.

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