4. Pellet cells by centrifugation at 3000 Â g for 10 min.
5. Wash cells three times at RT in 10% glycerol. Reduce the
volume each time; e.g., for 100 mL, wash one, 20 mL; wash
two, 10 mL; and wash three, 5 mL (see Note 15).
6. Resuspend in 1/50 to 1/100 original volume of 10% glycerol
(see Notes 16 and 17).
7. Add 0.5–5 μg salt-free DNA in no more than 5 μL volume to
0.2 mL mycobacterial suspension (see Notes 6 and 18).
8. Transfer to a 0.2-cm electrode gap electroporation cuvette (see
Note 19).
9. Place the cuvette in the electroporation chamber and subject to
one single pulse of 2.5 kV, 25 μF, with the pulse-controller
resistance set at 1000 Ω resistance (see Notes 20–22).
10. Recover cell suspension immediately into 10 mL of
7H9-OADC-Tw. Wash cuvette to recover all cells (see Note
23).
11. Incubate at 37
C for 16 h. This step allows expression of any
antibiotic-resistance gene carried on the DNA.
12. Harvest the cells by centrifugation at 3000 Â g for 10 min and
plate out suitable dilutions (to give 30–300 colonies per plate)
on 7H10-OADC plates and appropriate antibiotic (see Notes
24 and 25).
13. Incubate plates at 37
C until colonies become visible; this will
take approximately 3–4 weeks.
14. Count transformants to calculate transformation efficiency.
15. Streak out transformants onto 7H10-OADC plates plus selection (see Note 25).
16. Analyze transformants as required (see Note 26).
3.2 Electroporation
of M. smegmatis
1. Inoculate 5 mL Lemco broth with a loopful of mycobacteria.
Disperse the cells using a vortex (see Notes 9 and 27).
2. Incubate at 37
C with shaking (100 rpm) overnight.
3. Inoculate large-scale culture (100–500 mL in 250- to 1000mL conical flask) with a 1/100 dilution of the overnight culture and continue incubation at 37
C with shaking until
OD 600 ¼ 0.8–1.0 (usually between 16 and 24 h) (see Note 13).
4. Incubate cells on ice for 1.5 h (see Note 28).
5. Harvest cells by centrifugation at 3000 Â g for 10 min.
6. Wash cells three times in ice-cold 10% glycerol. Reduce the
volume each time; e.g., for 100 mL, wash one, 20 mL; wash
two, 10 mL; and wash three, 5 mL.
7. Resuspend in 1/10 to 1/100 original volume of ice-cold 10%
glycerol (see Notes 16 and 17).
Electroporation
277
5. Wash cells three times at RT in 10% glycerol. Reduce the
volume each time; e.g., for 100 mL, wash one, 20 mL; wash
two, 10 mL; and wash three, 5 mL (see Note 15).
6. Resuspend in 1/50 to 1/100 original volume of 10% glycerol
(see Notes 16 and 17).
7. Add 0.5–5 μg salt-free DNA in no more than 5 μL volume to
0.2 mL mycobacterial suspension (see Notes 6 and 18).
8. Transfer to a 0.2-cm electrode gap electroporation cuvette (see
Note 19).
9. Place the cuvette in the electroporation chamber and subject to
one single pulse of 2.5 kV, 25 μF, with the pulse-controller
resistance set at 1000 Ω resistance (see Notes 20–22).
10. Recover cell suspension immediately into 10 mL of
7H9-OADC-Tw. Wash cuvette to recover all cells (see Note
23).
11. Incubate at 37
C for 16 h. This step allows expression of any
antibiotic-resistance gene carried on the DNA.
12. Harvest the cells by centrifugation at 3000 Â g for 10 min and
plate out suitable dilutions (to give 30–300 colonies per plate)
on 7H10-OADC plates and appropriate antibiotic (see Notes
24 and 25).
13. Incubate plates at 37
C until colonies become visible; this will
take approximately 3–4 weeks.
14. Count transformants to calculate transformation efficiency.
15. Streak out transformants onto 7H10-OADC plates plus selection (see Note 25).
16. Analyze transformants as required (see Note 26).
3.2 Electroporation
of M. smegmatis
1. Inoculate 5 mL Lemco broth with a loopful of mycobacteria.
Disperse the cells using a vortex (see Notes 9 and 27).
2. Incubate at 37
C with shaking (100 rpm) overnight.
3. Inoculate large-scale culture (100–500 mL in 250- to 1000mL conical flask) with a 1/100 dilution of the overnight culture and continue incubation at 37
C with shaking until
OD 600 ¼ 0.8–1.0 (usually between 16 and 24 h) (see Note 13).
4. Incubate cells on ice for 1.5 h (see Note 28).
5. Harvest cells by centrifugation at 3000 Â g for 10 min.
6. Wash cells three times in ice-cold 10% glycerol. Reduce the
volume each time; e.g., for 100 mL, wash one, 20 mL; wash
two, 10 mL; and wash three, 5 mL.
7. Resuspend in 1/10 to 1/100 original volume of ice-cold 10%
glycerol (see Notes 16 and 17).
Electroporation
277