has been described in many variations in the last two editions of
Mycobacteria protocols [6, 22]. We use the CTAB method for its
enzymatic and chemical lysis of M. tuberculosis and NTM and to
remove cell components that could act as inhibitor molecules
downstream. CTAB in the presence of a high NaCl concentration
binds impurities such as polysaccharides and is coupled to chloroform–isoamyl DNA extraction and isopropanol precipitation
[23, 24]. Importantly, mechanical disruption is not used as this
would shear the mycobacterial chromosome. The protocol detailed
here will yield high-purity gDNA suitable for whole genome
sequencing and PCR methodologies.
2 Materials
2.1 Isolation of DNA
from M. tuberculosis
for Detection of
Plasmid Inserts
1. 10% (w/v) Tween-80: In a glass beaker, weigh 10 g of Tween80, add 80 mL of water. Mix on a stirrer with a stir bar until
dissolved. Adjust the volume with water to 100 mL. Filtersterilize using a 0.2 μm filter and store in the fridge for up to
1 year.
2. ADC (albumin, dextrose, and catalase supplement): Add
12.5 g BSA (stored at 4
C) to 200 mL Milli-Q water. Once
completely dissolved, add 5 g glucose, 0.0075 g catalase, and
2.125 g sodium chloride. Stir until completely dissolved and
filter-sterilize using a 0.2 μm filter. Store at 4
C.
3. Middlebrook 7H9 liquid medium: Dissolve 2.35 g of 7H9
(Difco) in 450 mL of Milli-Q water and add 1 mL glycerol
and autoclave to 121
C for 15 min. Cool the sterile medium to
55
C and add 50 mL ADC (albumin, dextrose, and catalase)
supplement (final 10%) and 2.5 mL of 10% Tween-80 (final
0.05%). Store at 4
C after addition of ADC.
4. OADC—oleic acid, albumin, dextrose, and catalase supplement (250 mL): Add 12.5 g BSA to 200 mL Milli-Q water;
once completely dissolved, add 5 g dextrose, 0.0075 g catalase,
0.125 g oleic acid, and 2.125 g sodium chloride and stir until
completely dissolved. Filter-sterilize using a 0.2 μm filter and
store at 4
C.
5. Middlebrook 7H11 solid medium (500 mL): Dissolve agar:
add 9.5 g 7H11 (Difco) powder and 2.5 mL 100% glycerol
(final 0.5%) in 450 mL of Milli-Q water and mix until
completely dissolved. Autoclave to 121
C and cool to
50–55
C. Add 50 mL OADC and 2.5 mL of 10% Tween-80
once ready to use. Set water bath to 98
C.
6. 1.5 mL screw cap O-ring tubes.
7. 100% chloroform.
8. Nuclease-free water.
62
Heena Jagatia and Daire Cantillon
Mycobacteria protocols [6, 22]. We use the CTAB method for its
enzymatic and chemical lysis of M. tuberculosis and NTM and to
remove cell components that could act as inhibitor molecules
downstream. CTAB in the presence of a high NaCl concentration
binds impurities such as polysaccharides and is coupled to chloroform–isoamyl DNA extraction and isopropanol precipitation
[23, 24]. Importantly, mechanical disruption is not used as this
would shear the mycobacterial chromosome. The protocol detailed
here will yield high-purity gDNA suitable for whole genome
sequencing and PCR methodologies.
2 Materials
2.1 Isolation of DNA
from M. tuberculosis
for Detection of
Plasmid Inserts
1. 10% (w/v) Tween-80: In a glass beaker, weigh 10 g of Tween80, add 80 mL of water. Mix on a stirrer with a stir bar until
dissolved. Adjust the volume with water to 100 mL. Filtersterilize using a 0.2 μm filter and store in the fridge for up to
1 year.
2. ADC (albumin, dextrose, and catalase supplement): Add
12.5 g BSA (stored at 4
C) to 200 mL Milli-Q water. Once
completely dissolved, add 5 g glucose, 0.0075 g catalase, and
2.125 g sodium chloride. Stir until completely dissolved and
filter-sterilize using a 0.2 μm filter. Store at 4
C.
3. Middlebrook 7H9 liquid medium: Dissolve 2.35 g of 7H9
(Difco) in 450 mL of Milli-Q water and add 1 mL glycerol
and autoclave to 121
C for 15 min. Cool the sterile medium to
55
C and add 50 mL ADC (albumin, dextrose, and catalase)
supplement (final 10%) and 2.5 mL of 10% Tween-80 (final
0.05%). Store at 4
C after addition of ADC.
4. OADC—oleic acid, albumin, dextrose, and catalase supplement (250 mL): Add 12.5 g BSA to 200 mL Milli-Q water;
once completely dissolved, add 5 g dextrose, 0.0075 g catalase,
0.125 g oleic acid, and 2.125 g sodium chloride and stir until
completely dissolved. Filter-sterilize using a 0.2 μm filter and
store at 4
C.
5. Middlebrook 7H11 solid medium (500 mL): Dissolve agar:
add 9.5 g 7H11 (Difco) powder and 2.5 mL 100% glycerol
(final 0.5%) in 450 mL of Milli-Q water and mix until
completely dissolved. Autoclave to 121
C and cool to
50–55
C. Add 50 mL OADC and 2.5 mL of 10% Tween-80
once ready to use. Set water bath to 98
C.
6. 1.5 mL screw cap O-ring tubes.
7. 100% chloroform.
8. Nuclease-free water.
62
Heena Jagatia and Daire Cantillon