Oneill Lab:DNA Extraction

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Michael J. O'Neill Lab University of Connecticut Department of Molecular and Cell Biology

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Protocol for extraction of DNA from tissue or embryos.

ProK Digestion

  1. Mix DNA extraction buffer
    • 98 μl ReagentB
    • 2 μl ProteinaseK
    • Mix fresh. 100 μl is enough for a small pea size chunk of tissue or one embryo
  2. Place small piece of tissue or embryo into a microfuge tube containing 100 μl of extraction buffer
  3. Incubate at 50°C overnight

PCI

  1. Prepare PCI mix
    1. One part tris-saturated phenol to one part 24:1 Chloroform:Isoamyl alcohol
    2. Shake thoroughly to make emulsion
  2. Add one volume of PCI to extracted sample
  3. Shake tubes for 10 seconds
  4. Centrifuge at max speed for 5 minutes
  5. Remove aqueous phase to a new tube
  6. Repeat as needed
  7. Add one volume 24:1 chloroform:isoamyl alcohol
  8. Shake tubes for 10 seconds
  9. Centrifuge at max speed for 5 minutes
  10. Remove aqueous phase to a new tube
  11. Continue to precipitation

Ethanol Precipitation

  1. Add 2 volumes 100% EtOH
  2. Add 1/10 volume 3M Sodium Acetate pH 5.0
  3. Centrifuge at max speed for 10 minutes
  4. Decant ethanol
  5. Add 150 μl 70% EtOH
  6. Centrifuge at max speed for 2 minutes
  7. Pipette out ethanol
  8. Airdry pellet
  9. Resuspend pellet in MilliQ water
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