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Bacterial Genomic DNA Isolation Protocol

Genomic DNA Isolation from Bateria Protocol

Preparation method for the isolation of geneomic DNA from bacteria using triton purification

  • Grow several colonies - large scale-15ml cultures.
  • Harvest the cells into a single eppendorf tube or a 15 ml disposable tube depending on the volume.
  • Resuspend pellet with 300ul STET buffer (900ul).
  • After resuspending add 30ul RNase/lysozyme mixture (100ul).
  • Boil pellet for 1 minute 15 seconds (one minute 45 seconds).
  • Spin in microfuge for 15 minutes.
  • Take supernatant and phenol extract with 150ul (500ul) STET- saturated phenol.
  • Spin and take supernatant. Add 1/10 volume 4M lithium chloride (autoclaved). Let sit on ice for 5-10 minutes.
  • Spin and take supernatant. Add equal volume isopropanol. RT for 5 minutes.
  • Spin. No pellet will be visible. Don't panic, DNA is stuck to side all the way up tube.
  • Important: Wash with 80% ethanol (95% will cause the residual Triton to precipitate)
  • Resuspend pellet in 50-200ul.
  • Lysozyme/ RNase mixture 10mg/ml lysozyme 1mg/ml RNase (use cheap grade (BMB) rather than RNase A , which is too expensive) 50mM Tris-HCl pH8.0 Store at -20oC in small aliquots. Do not refreeze after thawing.
  • STET 8% sucrose 5% Triton X-100 50mM Tris-HCl (pH8.0) 50mM EDTA pH 8.0 Filter sterilize. Store at 4oC

 

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