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Mechanical disruption of mycobacterial cells

Instrument: Precellys® 24 Dual

Sample: Mycobacteria obtained from cultures (note that pathogenic mycobacteria must be processed under appropriate biosafety containment).

Protocol:

• Add 0.1mm zirconia beads to 2mL tube
• Add 20 mg (wet wt) pellets of Mycobacteria re-suspended in phosphatebuffered saline (PBS; pH 7.4), and heat inactivated at 95°C for 60 min.
• Run the Precellys at 6800rpm, 3 x 30sec 
• Perform phenol-chloroform DNA extraction and chloroform purification.

Results:

Mechanical disruption of the mycobacterial cell wall, after incubation with 4% SDS (final concentration), was found to be crucial for sufficient and satisfactory yields.

DNA yield achieved when testing both combinations of DNA extraction procedures (chemical lysis only vs chemical and mechanical disruption)