TABLE 2.

Optimized protocol for preparation of mycobacterial DNA

Process and step	Vol (μl)
Cell wall disruption and digestion
Transfer 20-mg (wet wt) pellet or environmental sample into a 1.5-ml screw-cap tube and wash with PBS if needed	∼10
Resuspend in lysis buffer (15% sucrose, 0.05 M Tris [pH 8.5], 0.05 M EDTA)	300
Add lysozyme (stock, 100 mg/ml; end concn, 10 mg/ml):	50
Incubate for 1 h at 37°C
Add SDS (stock, 20%; end concn, 4%)	100
Add PK (stock, 2.5 mg/ml; end concn, 0.2 mg/ml)	40
Total	500
Incubate for 1 h at 37°C
Incubate for 5 min at 70°C
Add matrix material (vol of ∼150 μl glass or zirconia beads of 0.1-mm diam) and homogenize with mechanical treatment device
Centrifuge at 14,000 rpm for 2 min
Transfer supernatant to 1.5-ml reaction tube
Phenol-chloroform extraction and EtOH precipitation
Add phenol-chloroform-isoamyl alcohol (Tris saturated)	500
Vortex, centrifuge at 14,000 rpm for 5 min
Collect supernatant
Repeat phenol-chloroform extraction step
Add chloroform-isoamyl alcohol	500
Vortex, centrifuge at 14,000 rpm for 5 min
Collect supernatant	∼200
Add 3 M sodium acetate	20
Add 100% EtOH	500
Freeze for >30 min at −70°C, centrifuge at 14,000 rpm for 30 min at 4°C
Wash with 70% EtOH	700
Centrifuge at 14,000 rpm for 10 min at 4°C
Repeat EtOH wash step
Dry at 50°C, resuspend in nuclease-free H2O	100
Quality control
Measure DNA concn and purity with a spectrophotometer
DNA concn: >100 ng/μl; protein: 260/280 nm, >1.8; detergent: 260/230 nm, >1.8
Monitor quality on 1% agarose gel