5. 1.5 mL screw-cap tubes with O-ring.
6. Bead beater (MP Biotechnologies).
7. Breaking buffer: 50 mM Tris-Cl pH 8.0, 10 mM EDTA,
100 mM NaCl, autoclave at 121
C for 15 min. RNase
(DNase-free) is added to a final concentration of 200 μg/mL
immediately before use.
8. SDS solution: Prepare 10% (w/v) SDS solution, filter-sterilize.
9. 10 mg/mL proteinase K.
10. Phenol/chloroform/isoamyl alcohol—25:24:1: Prepared or
purchased which is already made into a 25:24:1 mix.
11. 3 M sodium acetate.
12. 100% molecular-grade isopropanol.
13. 100% molecular-grade ethanol.
14. 10,000 U/mL BamHI HF with CutSmart buffer (NEB).
15. TE buffer: 10 mM Tris-Cl, pH 8.0, 1 mM EDTA, sterilize by
autoclaving.
2.4 Genomic
Extractions from M.
tuberculosis and
Nontuberculous
Mycobacteria (NTM)
Using CTAB Extraction
Method
1. M. tuberculosis and nontuberculous mycobacteria cultures (see
Subheading 2.1, items 1–3, note culturing of NTM is not
described in this chapter, but the same protocol can be used
with NTM cultures).
2. 1.5 mL O-ring screw-capped tubes.
3. Parafilm.
4. Water bath set to 37
C; 65
C.
5. Heating block at 65
C.
6. DNA LoBind 1.5 mL tubes (Eppendorf).
7. Sterile TE buffer (10 mM Tris-Cl pH 8.0; 1 mM EDTA
pH 8.0).
8. 10 mg/mL lysozyme.
9. 10 mg/mL RNase A.
10. CTAB/NaCl 10% (w/v) CTAB; 700 mM NaCl buffer: Dissolve 8.2 g of NaCl in 40 mL Milli-Q water, followed by 20 g
CTAB and heat in a water bath until all components have
dissolved. Make up to 200 mL final volume and autoclave at
121
C for 15 min. Assign shelf life of 6 months if stored at
4
C.
11. 10% sodium dodecyl sulphate.
12. 20 mg/mL proteinase K.
13. 5 M NaCl solution: Dissolve 5.84 g NaCl in 20 mL of Milli-Q
water by heating in a 65
C water bath. Assign a shelf life of
6 months if stored at ambient temperature.
64
Heena Jagatia and Daire Cantillon
6. Bead beater (MP Biotechnologies).
7. Breaking buffer: 50 mM Tris-Cl pH 8.0, 10 mM EDTA,
100 mM NaCl, autoclave at 121
C for 15 min. RNase
(DNase-free) is added to a final concentration of 200 μg/mL
immediately before use.
8. SDS solution: Prepare 10% (w/v) SDS solution, filter-sterilize.
9. 10 mg/mL proteinase K.
10. Phenol/chloroform/isoamyl alcohol—25:24:1: Prepared or
purchased which is already made into a 25:24:1 mix.
11. 3 M sodium acetate.
12. 100% molecular-grade isopropanol.
13. 100% molecular-grade ethanol.
14. 10,000 U/mL BamHI HF with CutSmart buffer (NEB).
15. TE buffer: 10 mM Tris-Cl, pH 8.0, 1 mM EDTA, sterilize by
autoclaving.
2.4 Genomic
Extractions from M.
tuberculosis and
Nontuberculous
Mycobacteria (NTM)
Using CTAB Extraction
Method
1. M. tuberculosis and nontuberculous mycobacteria cultures (see
Subheading 2.1, items 1–3, note culturing of NTM is not
described in this chapter, but the same protocol can be used
with NTM cultures).
2. 1.5 mL O-ring screw-capped tubes.
3. Parafilm.
4. Water bath set to 37
C; 65
C.
5. Heating block at 65
C.
6. DNA LoBind 1.5 mL tubes (Eppendorf).
7. Sterile TE buffer (10 mM Tris-Cl pH 8.0; 1 mM EDTA
pH 8.0).
8. 10 mg/mL lysozyme.
9. 10 mg/mL RNase A.
10. CTAB/NaCl 10% (w/v) CTAB; 700 mM NaCl buffer: Dissolve 8.2 g of NaCl in 40 mL Milli-Q water, followed by 20 g
CTAB and heat in a water bath until all components have
dissolved. Make up to 200 mL final volume and autoclave at
121
C for 15 min. Assign shelf life of 6 months if stored at
4
C.
11. 10% sodium dodecyl sulphate.
12. 20 mg/mL proteinase K.
13. 5 M NaCl solution: Dissolve 5.84 g NaCl in 20 mL of Milli-Q
water by heating in a 65
C water bath. Assign a shelf life of
6 months if stored at ambient temperature.
64
Heena Jagatia and Daire Cantillon