11. Kanamycin sulfate: prepare as 50 mg/mL stock, filter-sterilize,
store at À20
C. Use at final concentration of 10–30 μg/mL in
selective agar plates.
12. Hygromycin B: 50 mg/mL stock in phosphate-buffered saline;
store at 4
C in the dark. Use at final concentration of
50–100 μg/mL in selective agar plates.
2.2 Electroporation
of M. smegmatis
1. Mycobacterium smegmatis strain mc
2 155 (see Note 8).
2. Tween-80: Prepare as a 20% w/v stock, filter-sterilize through
a 0.2-μm membrane and store at 4
C (see Note 2).
3. Lemco broth: 5 g/L peptone, 5 g/L Lemco powder, 5 g/L
NaCl, 5 mL/L of 10% w/v Tween-80. Autoclave (see Note 9).
4. 2 M glycine, autoclave.
5. 10% w/v glycerol; sterilize by autoclaving or filtration. Store at
4
C.
6. Electroporation apparatus with pulse controller (see Note 5).
7. Electroporation cuvettes; 0.2-cm gap electrodes.
8. DNA solution: 0.2–1 mg/mL. This should be free from salts,
enzymes and other substances (see Note 6).
9. Lemco agar: 5 g/L peptone, 5 g/L Lemco powder, 5 g NaCl,
15 g/L agar. Autoclave. Add antibiotics if required. Pour plates
and use within 1 week (see Notes 7 and 9).
10. Kanamycin sulfate: prepare as 50 mg/mL stock, filter-sterilize,
store at À20
C. Use at final concentration of 10–30 μg/mL in
selective agar plates.
11. Hygromycin B: 50 mg/mL stock in phosphate-buffered saline;
store at 4
C in the dark. Use at final concentration of
50–100 μg/mL in selective agar plates.
3 Methods
3.1 Electroporation
of M. tuberculosis
Caution: M. tuberculosis is a human pathogen; therefore, appropriate containment facilities should be used for all procedures (see
Note 1).
1. Inoculate 10 mL 7H9-OADC-Tw with a loopful of M. tuberculosis, vortex to disperse cells, and incubate the starter culture
at 37
C for 10–15 days (see Notes 10 and 11).
2. Inoculate 100 mL 7H9-OADC-Tw in a roller bottle with
1–10 mL of starter culture and continue incubation at 37
C
with rolling at 100 rpm for 5–7 days (see Notes 12 and 13).
3. Add 0.1 volumes 2 M glycine (final concentration 1.5% w/v)
16–24 h before harvesting the cells (see Note 14).
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Tanya Parish
store at À20
C. Use at final concentration of 10–30 μg/mL in
selective agar plates.
12. Hygromycin B: 50 mg/mL stock in phosphate-buffered saline;
store at 4
C in the dark. Use at final concentration of
50–100 μg/mL in selective agar plates.
2.2 Electroporation
of M. smegmatis
1. Mycobacterium smegmatis strain mc
2 155 (see Note 8).
2. Tween-80: Prepare as a 20% w/v stock, filter-sterilize through
a 0.2-μm membrane and store at 4
C (see Note 2).
3. Lemco broth: 5 g/L peptone, 5 g/L Lemco powder, 5 g/L
NaCl, 5 mL/L of 10% w/v Tween-80. Autoclave (see Note 9).
4. 2 M glycine, autoclave.
5. 10% w/v glycerol; sterilize by autoclaving or filtration. Store at
4
C.
6. Electroporation apparatus with pulse controller (see Note 5).
7. Electroporation cuvettes; 0.2-cm gap electrodes.
8. DNA solution: 0.2–1 mg/mL. This should be free from salts,
enzymes and other substances (see Note 6).
9. Lemco agar: 5 g/L peptone, 5 g/L Lemco powder, 5 g NaCl,
15 g/L agar. Autoclave. Add antibiotics if required. Pour plates
and use within 1 week (see Notes 7 and 9).
10. Kanamycin sulfate: prepare as 50 mg/mL stock, filter-sterilize,
store at À20
C. Use at final concentration of 10–30 μg/mL in
selective agar plates.
11. Hygromycin B: 50 mg/mL stock in phosphate-buffered saline;
store at 4
C in the dark. Use at final concentration of
50–100 μg/mL in selective agar plates.
3 Methods
3.1 Electroporation
of M. tuberculosis
Caution: M. tuberculosis is a human pathogen; therefore, appropriate containment facilities should be used for all procedures (see
Note 1).
1. Inoculate 10 mL 7H9-OADC-Tw with a loopful of M. tuberculosis, vortex to disperse cells, and incubate the starter culture
at 37
C for 10–15 days (see Notes 10 and 11).
2. Inoculate 100 mL 7H9-OADC-Tw in a roller bottle with
1–10 mL of starter culture and continue incubation at 37
C
with rolling at 100 rpm for 5–7 days (see Notes 12 and 13).
3. Add 0.1 volumes 2 M glycine (final concentration 1.5% w/v)
16–24 h before harvesting the cells (see Note 14).
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Tanya Parish