3. The medium used for growth of mycobacteria for electroporation is not critical. A variety of different recipes are used, the
most common liquid being Middlebrook 7H9 which can be
supplemented with either OADC or ADC (no oleic acid). The
base medium can be stored before addition of the supplement.
The most common solid medium is Middlebrook 7H10 agar
which is supplemented with OADC.
4. ADC and OADC supplements are heat-labile and should be
added to the base medium after cooling. We have found best
results when supplemented medium is stored for less than
1 week.
5. There are many different electroporation devices and cuvettes
available commercially; any apparatus that can deliver highvoltage pulses can be used. Cuvette capacity depends on the
height and gap; therefore, the maximum volume of cell suspension can vary from 50 μL to 400 μL. The volume of cell
suspension used does not affect the efficiency.
6. Insure that the DNA for transformation is free from salts and
other contaminants, ethanol precipitation and washing with
70% ethanol can be used to clean up DNA, which should
preferably be dissolved in sterile deionized, distilled water.
7. Kanamycin and hygromycin are the most commonly used
selectable markers, but others are available including resistance
markers for gentamicin, streptomycin, and apramycin
[4, 11]. Ampicillin is not used, since mycobacteria are naturally
resistant to beta-lactams. Chloramphenicol cannot be used for
direct selection owing to the high rate of spontaneous
mutations.
8. We recommend the use of M. smegmatis mc
2 155, a derivative
of M. smegmatis ATCC607 [19]. This strain has much higher
transformation efficiencies than the parental strain (10
4 to 10
5
per μg of DNA) and is the most commonly used in laboratory
settings. This strain also has the advantage that it does not
possess a restriction and modification system.
9. We have also used Middlebrook 7H9 medium supplemented
with ADC or OADC and Tween-80 to culture M. smegmatis.
However, the OADC supplements tend to be expensive and
since M. smegmatis will grow robustly in Lemco (or Luria
Bertani) medium, we recommend its use.
10. Mycobacteria are relatively slow-growing organisms; the fastgrowing species have a generation of 2–3 h and the slowgrowing species of around 20 h. This can lead to a problem
with contamination of cultures because many common contaminants have a much quicker doubling time and will rapidly
outgrow mycobacteria. It is extremely important to maintain a
good aseptic technique, especially with slow-growers. It is
Electroporation
279
most common liquid being Middlebrook 7H9 which can be
supplemented with either OADC or ADC (no oleic acid). The
base medium can be stored before addition of the supplement.
The most common solid medium is Middlebrook 7H10 agar
which is supplemented with OADC.
4. ADC and OADC supplements are heat-labile and should be
added to the base medium after cooling. We have found best
results when supplemented medium is stored for less than
1 week.
5. There are many different electroporation devices and cuvettes
available commercially; any apparatus that can deliver highvoltage pulses can be used. Cuvette capacity depends on the
height and gap; therefore, the maximum volume of cell suspension can vary from 50 μL to 400 μL. The volume of cell
suspension used does not affect the efficiency.
6. Insure that the DNA for transformation is free from salts and
other contaminants, ethanol precipitation and washing with
70% ethanol can be used to clean up DNA, which should
preferably be dissolved in sterile deionized, distilled water.
7. Kanamycin and hygromycin are the most commonly used
selectable markers, but others are available including resistance
markers for gentamicin, streptomycin, and apramycin
[4, 11]. Ampicillin is not used, since mycobacteria are naturally
resistant to beta-lactams. Chloramphenicol cannot be used for
direct selection owing to the high rate of spontaneous
mutations.
8. We recommend the use of M. smegmatis mc
2 155, a derivative
of M. smegmatis ATCC607 [19]. This strain has much higher
transformation efficiencies than the parental strain (10
4 to 10
5
per μg of DNA) and is the most commonly used in laboratory
settings. This strain also has the advantage that it does not
possess a restriction and modification system.
9. We have also used Middlebrook 7H9 medium supplemented
with ADC or OADC and Tween-80 to culture M. smegmatis.
However, the OADC supplements tend to be expensive and
since M. smegmatis will grow robustly in Lemco (or Luria
Bertani) medium, we recommend its use.
10. Mycobacteria are relatively slow-growing organisms; the fastgrowing species have a generation of 2–3 h and the slowgrowing species of around 20 h. This can lead to a problem
with contamination of cultures because many common contaminants have a much quicker doubling time and will rapidly
outgrow mycobacteria. It is extremely important to maintain a
good aseptic technique, especially with slow-growers. It is
Electroporation
279