Preparation of genomic DNA from adult flies


You need:

  • 200 flies
  • mortar and pistle
  • liquid nitrogen
  • centrifugation vials (15 ml)
  • 10 ml Dounce homogeniser with pestle
  • Homogenisation buffer (10 mM Tris/HCl [pH7,5], 60 mM NaCl, 10 mM EDTA)
  • Proteinase K (10 mg/ml)
  • 10% SDS
  • 3 M NaCl
  • phenol-chloroform-IAA (50:49:1)
  • chlorophorm-IAA (24:1).
  • glass rods (made from 50µl glass capillaries)
  • ethanol, ethanol (80%)
  • microfuge, horizontal shaker
  1. Anesthesise flies (about 200) on ice, transfer them to a mortar with liquid nitrogen (do not let the flies wake up and fly away) and grind with a pistle until you get a homogenous powder of flies. Transfer the powder to a cooled Dounce homogeniser containing 5 ml of homogenisation buffer
  2. Grind with a few strokes of the pestle to release cells and nuclei from the caracasses of the flies. Centrifuge to remove debris at 1000 rpm for 1 min (but no more!).
  3. Transfer the supernatant with a pipette to a centrifugation tube, spin in the Sorvall at 8000 rpm for 5 min.
  4. Discard the supernatant (cytoplasm with RNA), collect the pellet (nuclei) and resuspend in 0,5 ml homogenisation buffer, transfer to a 2 ml reaction vial.
  5. Add proteinase K to a final concentration of 100 µg/ml and mix. Add 50 µl of 10% SDS and mix by swirling and rocking.
  6. incubate at 37°C for 45 to 60 min.
  7. Add 0.5 ml phenol/chloroform (wearing gloves!) and place tube on a horizontal shaker for 5 min. (don't vortex - it would share large genomic DNA to pieces; make sure the Eppendorf tube is firmly sealed with parafilm)
  8. Spin 3 min in microfuge at full speed, and transfer supernatant into a new Eppendorf tube, use the P1000 pipetman; if the DNA is "gluey", use a blue tip with a wider opening).
  9. Repeat step 5&6 with phenol-chloroform-IAA (50:49:1)
  10. Repeat step 5&6 with chlorophorm-IAA (24:1).
  11. Fill one Eppendorf tube with 1.5 ml 80% ethanol, another one with 100% ethanol (for wash steps in 12.).
  12. Add NaCl to a final concentration of 200 mM, mix. Add two volumes of 100% ethanol. Mix by gentle swirling. The DNA should appear at the interface as a clump. After a white thread-like precipitate formed, use a Pasteur pipette (glass hook) to transfer the precipitate to the tube with 80 % ethanol.
  13. Wash the precipitate in 80% ethanol and then 100% ethanol.
  14. Air-dry the precipitate to remove all ethanol and dislodge pellet into 500 µl TE buffer.
  15. Rotate the tube on a horizontal shaker to dissolve the DNA.
  16. Take the OD260 from a 5 µl sample. A typical preparation yields 100-500 µg DNA. Store DNA at 4°C and clearly mark the number on the tube.