Bacterial Genomic DNA Isolation Protocol

Written by Super User. Posted in DNA

Method for the preparation of isolation of genomic DNA from bacteria.

Genomic DNA Isolation from Bateria Protocol

Preparation method for the isolation of genomic 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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