Team:Calgary/Notebook/Protocols/Process24

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Genomic DNA Isolation from Pseudomonas spp.

This protocol is used to isolate genomic DNA (gDNA) from Pseudomonas spp. (and any gram negative bacteria), and is adapted from the Chen & Kuo (1993) publication in Nucleic Acids Research.

Reagents

  • Saturated cell culture
  • Lysis buffer (40 mM Tris-acetate pH 7.8, 20 mM sodium-acetate, 1 mM EDTA, 1% SDS)
  • 5M NaCl
  • Chloroform
  • 100% ethanol
  • 70% ethanol
  • ddH2O

    • Protocol

    1. Centrifuge 1.5mL of a saturated culture for 3 min at 12000 rpm.
    2. Decant the supernatant, and resuspend the cell pellet in 200µL of lysis buffer. Mix by vigorous pipetting.
    3. Add 66µL of 5M NaCl to remove proteins and cell debris. Mix well.
    4. Centrifuge at 12000rpm for 10 min at 4°C.
    5. Transfer the clear supernatant to a new tube, and precipitate DNA by adding 100% ethanol, and incubating at -20°C for at least 1 hour.
    6. Centrifuge for 10 minutes at 4°C.
    7. Decant the supernatant and wash the pellet with 70% ethanol twice.
    8. Dry the pellet in a speed vac or over a clean piece of paper towel for 30 min.
    9. Resuspend the pellet in 50µL of ddH2O and measure the DNA concentration using Nanodrop.

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