Isolation Purification and Separation

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Authors: Radoslaw Kamil Ejsmont 

Introduction

This is a quick and robust protocol for isolation of High Molecular Weight genomic DNA from Drosophila embryos. We have used DNA isolated using this protocol for fosmid genomic library production.

Procedure

  1. Collect embryos for 24h. Optional: Let them age for up to 12h at room temperature.
  2. Decorionate embryos for 2 minutes in 100% bleach fluid (2% sodium perchloride).
  3. Wash embryos with 1 x PBS.
  4. Wash embryos with 1 x PBT.
  5. Transfer embryos into a bottle containing 1 volume of PBS and 1 volume of n- Heptane. Use 20 ml of PBS per 1 ml of embryos. Mix by briefly shaking the bottle.
  6. Remove PBS (lower phase). Leave the interphase intact.
  7. Add 1 volume of methanol and shake vigorously by hand for 1 minute.
  8. Remove n-heptane and interphase.
  9. Transfer embryos into the Falcon tube (15ml or 50ml depending on the sample volume) and wash twice with 1 volume of methanol.
  10. Remove methanol.
  11. Wash embryos with 1 volume of PBS.
  12. Add 1 volume of lysis buffer (50 mM Tris-HCl pH 8.0, 100 mM EDTA, 100 mM NaCl, 0.5% SDS, 5 mg/ml Proteinase K, 100 mg/ml RNAse A). Lyse for 2-3 hours at 55°C. Gently mix by inverting the tube every 15 minutes.
  13. Centrifuge at 4000g for 15 minutes. Transfer supernatant to a new Falcon tube. Optional: Remove 200 μl for quality analysis.
  14. Add 1 volume of Phenol:Chloroform:Isoamyl alcohol. Incubate on a rotating wheel for 1 hour at 4°C.
  15. Centrifuge at 4000g for 10 minutes. Transfer aqueous (upper) phase to a new Falcon tube.
  16. Repeat steps 14–15.
  17. Add 1 volume of Chloroform:Isoamyl alcohol. Incubate on a rotating wheel for 1 hour at 4°C.
  18. Centrifuge at 4000g for 10 minutes. Transfer aqueous (upper) phase to a new Falcon tube.
  19. Add 0.05 volume of 3.0 M KAc. Mix by gently inverting the tube.
  20. Add 0.7 volume of isopropanol. Incubate on a rotating wheel for 30 minutes at 4°C.
  21. Centrifuge at 6000g for 15 minutes. Remove supernatant.
  22. Wash the pellet twice with 1 volume of 70% ethanol.
  23. Air-dry the pellet for 10 minutes at room temperature.
  24. Dissolve the pellet in 1 x TE prewarmed to 55°C. Store DNA at 4°C.

Associated Publications

A toolkit for high-throughput, cross-species gene engineering in Drosophila, Radoslaw K Ejsmont, Mihail Sarov, Sylke Winkler, Kamil A Lipinski, and Pavel Tomancak, Nature Methods 6 (6) 435 - 437 24/05/2009 doi:10.1038/nmeth.1334

Author information

Radoslaw Kamil Ejsmont, MPI-CBG

Correspondence to: Radoslaw Kamil Ejsmont ([email protected])

Source: Protocol Exchange (2009) doi:10.1038/nprot.2009.107. Originally published online 27 May 2009.

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