Wednesday, May 6, 2009

Bacterial DNA Extraction for Pulsed-field Gel Electrophoresis

Pulsed-field gel electrophoresis (PFGE) is a method used to separate large DNA fragments such as those obtained after digestion with restriction endonucleases that cut infrequently. To avoid possible risks of shearing bacterial DNA during the extraction and digestion steps, bacterial cells are immobilized prior to processing by incorporation in agarose gel. DNA extraction for PFGE is characterized by the need to prolong contact between agarose plugs and the lysis solution that must be distributed throughout the gel. However, the duration of DNA preparation has been shortened since the initial description of the method. The method described in this posting works well with Gram-positive and Gram-negative rods of clinical interest.

Materials that are needed
  1. TE buffer: 10 mM Tris-HCl, 1 mM EDTA, pH 8.
  2. Lysis buffer: 6 mMTris-HCl, 1M NaCl, 0.1 M EDTA, 0.5% sodium N-lauroyl-sarcosine, pH 8.

    Other detergents such as sodium deoxycholate (0.2%) and Brij 58 (0.5%) (Sigma Chemicals, St. Louis, MO) may be added.

  3. Lyzozyme solution: 85 mg/mL in sterile water.
    Lysostaphine solution: 1.000 U/mL in sterile water. The stock solution is stored in aliquots frozen at -20oC.
  4. Low melting agarose: 2% in sterile water.
  5. Sterile petri dishes 55 mm in diameter.
  6. Digestion solution: EDTA 0.5 M, 1% sodium N-lauroyl sarcosine, proteinase K 2 mg/mL, pH 8.
  7. PMSF (phenylmethysulfonyl-fluoride): 40 mg/mL in isopropanol. PMSF is harmful, so avoid contact with mucous membranes and eyes. It should not be inhaled and the solution must be handled in a chemical hood.

The Method is

Quantities are for the preparation of five plugs of 200 microliters each.
  1. Grow bacteria in appropriate liquid medium overnight and shake if necessary.
  2. Pellet cells by centrifugation at 2000g and wash them twice with TE buffer. The amount of cells required is equivalent to that found in 5 mL of broth culture with an optical density of 1.0 at 600 nm.
  3. Resuspend cells in 500 microliters of TE buffer.
  4. Add 75 microliters of lyzozyme solution. For staphylococci, also add 15 microliters of lysostaphine.
  5. Add 750 microliters of melted (55oC) 2% low melting agarose.
  6. Mix thoroughly and pipet the agarose-cell suspension into the wells of a plug mold. Allow agarose plugs to solidify for at least 10 min at 4oC.
  7. Gently remove the plugs from the mold and transfer them into a sterile petri dish containing 10 mL of lysis buffer. Incubate for 2 h at 37oC for Gram-negative bacteria and for 4 h for Gram-positive bacteria.
  8. Remove the lysis buffer and replace it with 10 mL of the digestion solution. Incubate the petri dishes at 50oC overnight. Close them firmly with parafilm to avoid evaporation of the solution.
  9. Cool the petri dish at 4oC. Transfer the agarose plugs into a new petri dish containing 10 mL of TE buffer. Wash the plugs three times in 10 mL of TE buffer with gentle shaking.
  10. Remove the last 10 mL of washing buffer, add 5 mL of TE buffer.
  11. Add 60 microliters of PMSF isopropanol solution twice 30 min apart and incubate at 50oC. Rinse plugs three times in TE buffer.

    PMSF is used to inhibit residual proteinase K activity. It is insoluble in aqueous solution where its half-life is 60 min. This step is not included in many protocols which use lower proteinase K concentrations.

  12. Store plugs in 0.2 M EDTA at 4oC. DNA prepared and stored is stable for several months. Plugs have to be rinsed three times for 10 min each in 10 mL of TE buffer to eliminate EDTA before digestion of DNA with restriction enzymes.


Reference: Number 12 on References