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Seven treatments for bacterial sample materials with MagNA Pure Bacteria Lysis Buffer

 

  1. MagNA Lyser treatment (optional automated mechanical homogenization)
  2. Liquefaction (optional step for very viscous samples, such as BAL, sputum)
  3. Centrifugation (optional step for large volume liquid samples that have low or unknown bacterial loads, such as urine, CSF, BAL, aspirates)
  4. Addition of Bacteria Lysis Buffer
  5. Enzymatic digestion (optional step to enhance lysis of certain bacteria, such as gram-positive species)
  6. Proteinase K digestion
  7. Boiling (highly recommended for inactivating pathogenic organisms in the sample)

 

MagNA Lyser treatment (optional automated mechanical homogenization)

  • Enhances lysis of especially gram-positive bacterial species.
  • Very useful for samples that contain particles, such as stool.
  • Transfer sample (up to 500 μl) to a MagNA Lyser Green Beads tube.
  • Place the tube in the MagNA Lyser Instrument.
  • Homogenize for 30 seconds at 6,000 rpm.
  • Cool samples for 1 minute in the MagNA Lyser Rotor Cooling Block.
  • Centrifuge 5 minutes at 17,000 x g (at +15 to +25°C).
  • Transfer 400 μl of the lysate supernatant into the sample tube.

Note: For some types of sample, you may improve DNA yield by performing this homogenization step first, before adding Bacteria Lysis Buffer.
 

Liquefaction (optional step for very viscous samples, such as BAL, sputum)

  • Prepare a fresh DTT stock solution (e.g., 5x concentrated = 0.75%).
  • Adjust the final concentration of DTT in the sample to 0.15% by adding appropriate amounts of DTT stock solution.
  • Incubate the sample by shaking for 30 minutes at +37°C, until it can be easily pipetted.

 

Centrifugation (optional step for large volume liquid samples that have low or unknown bacterial loads, such as urine, CSF, BAL, aspirates)

  • Centrifuge the sample for up to 10 minutes at 20,000 x g to concentrate the bacterial cells in the pellet.
  • Discard most of the supernatant leaving only 200 μl.
  • Resuspend the cells in this remaining liquid, then process this concentrate.
    This centrifugation step allows a dilute sample that originally contains several milliliters of liquid to be processed in a single isolation, ensuring maximum DNA yield.

 

Addition of Bacteria Lysis Buffer

  • Add 180 μl of MagNA Pure Bacteria Lysis Buffer (BLB) to 200 μl of sample.
  • If the sample volume is less than 200 μl, add enough BLB for a final volume of 380 μl.
  • When the MagNA Lyser is used to homogenize the sample (see above), increase the volume of both the sample and BLB to 250 μl each. (Final volume, BLB + sample = 500 μl)
  • Mix well

 

Enzymatic digestion (optional step to enhance lysis of certain bacteria, such as gram-positive species)

  • Depending on the type of bacteria to be lysed, prepare one of the following enzyme cocktails: Enzyme Cocktail I: N-acetylmuramidase (0.625 mg/ml = 2500 U/ml) and beta-1,3-glucanase (Zymolyase) (0.25 mg/ml = 500 U/ml); dissolved in 50% glycerol/50 mM Tris-acetat, pH 6.7. [This cocktail lyses a broad spectrum of gram-positive bacteria.] Enzyme Cocktail II: lysozyme (100 mg/ml = 50 kU/ml) and lysostaphin (5 mg/ml = 5 kU/ml); dissolved in 5 % glycerol/PBS, pH 7.5. [This cocktail is especially useful for lysis of Staphylococcus spec.]
  • Add 10 μl (per 400 μl total sample volume of the enzyme cocktail) to the BLB/sample mixture.
    Note: You can premix Enzyme Cocktail and BLB and add them both to the sample in a single step.
  • Incubate at +37°C for 10 to 30 minutes.

 

Proteinase K digestion

  • Add 20 μl of Proteinase K to the mixture and mix thoroughly.
    Note: If you do not use an enzyme cocktail, premix Proteinase K and BLB and add them to the sample in a single step.

When the MagNA Lyser is used to homogenize the sample (see above), increase the volume of both the sample and BLB/Proteinase K to 250 μl each. (Final volume, BLB + Proteinase K + sample = 500 μl)

  • Incubate for 10 minutes at +65°C (e.g., BALs) or overnight at room temperature such as biopsies, solid tissue until the sample is completely dissolved and can be resuspended.

 

Boiling

Note: Roche strongly recommends performing this step to inactivate pathogenic organisms in the sample. It may also enhance lysis of the cell wall of some bacteria species. To prevent leakage, perform this step in screw-capped reaction tubes.

  • Incubate samples at +95°C for 10 minutes.
  • Recentrifuge briefly to collect the complete sample volume at the bottom of the tube.
  • Allow samples to cool down or chill on ice, then transfer the cooled sample to the MagNA Pure Compact Sample Tube.