background lawn of sensitive cells. This is owing to aggregation, which protect some cells from the effects of the antibiotic.
Because slow-growing organisms take up to 4 weeks to form
colonies from single cells, it is important to pour plates thickly
and to wrap them securely in parafilm or sealable bags to
prevent drying out during the long incubation period. Cycloheximide can be added to plates (100 μg/mL) to prevent
fungal contamination, which is possible during such long incubation times. The long incubation period also means that
antibiotic-containing plates should be freshly poured for each
experiment; this will minimize the loss of antibiotic activity.
25. Transformants may be inoculated directly into liquid medium,
but we find that they grow more rapidly if they are first streaked
out to get good growth on plates. For most applications, three
transformants should be streaked and analyzed. It is important
to check the identity of extrachromosomal plasmids after transformation, since deletions and rearrangements are common.
26. Spontaneous kanamycin resistance can often be a problem with
M. tuberculosis, owing to mutations in the rrn operon, of which
slow-growing species possess only one [17]. A control electroporation with no DNA to check for the frequency of such
mutants can be included. Problems of spontaneous resistance
will apply to all antibiotics that act on rrn operon (e.g.,
streptomycin).
27. We routinely generate starter cultures of M. smegmatis using
5 mL of medium in a 20-mL conical bottom universal tube and
incubating overnight with shaking. Other methods of culture
using small stirred cultures in glass tubes, rolling/rotating
tubes, or tissue culture flasks are equally acceptable. Selection
of the culture method will depend on the equipment and
resources available. Cultures can also be started from frozen
stocks.
28. For fast-growing species, once cultures have reached the
required stage of growth, they should be removed from the
incubator and incubated on ice for 1.5 h prior to harvesting.
This results in a fourfold increase in transformation efficiency.
Longer incubations on ice result in reduced efficiency, probably
owing to increased cell lysis. This may also increase the possibility of arcing during the pulse delivery.
29. Competent cells that have been thawed from frozen should be
harvested and resuspended in fresh 10% glycerol prior to use to
remove any lysed bacteria. For M. smegmatis, we have found
that frozen cells are more competent than freshly prepared
cells.
30. It is important to have an even cell suspension for electroporation because any clumping of cells will lead to arcing and
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Tanya Parish
Because slow-growing organisms take up to 4 weeks to form
colonies from single cells, it is important to pour plates thickly
and to wrap them securely in parafilm or sealable bags to
prevent drying out during the long incubation period. Cycloheximide can be added to plates (100 μg/mL) to prevent
fungal contamination, which is possible during such long incubation times. The long incubation period also means that
antibiotic-containing plates should be freshly poured for each
experiment; this will minimize the loss of antibiotic activity.
25. Transformants may be inoculated directly into liquid medium,
but we find that they grow more rapidly if they are first streaked
out to get good growth on plates. For most applications, three
transformants should be streaked and analyzed. It is important
to check the identity of extrachromosomal plasmids after transformation, since deletions and rearrangements are common.
26. Spontaneous kanamycin resistance can often be a problem with
M. tuberculosis, owing to mutations in the rrn operon, of which
slow-growing species possess only one [17]. A control electroporation with no DNA to check for the frequency of such
mutants can be included. Problems of spontaneous resistance
will apply to all antibiotics that act on rrn operon (e.g.,
streptomycin).
27. We routinely generate starter cultures of M. smegmatis using
5 mL of medium in a 20-mL conical bottom universal tube and
incubating overnight with shaking. Other methods of culture
using small stirred cultures in glass tubes, rolling/rotating
tubes, or tissue culture flasks are equally acceptable. Selection
of the culture method will depend on the equipment and
resources available. Cultures can also be started from frozen
stocks.
28. For fast-growing species, once cultures have reached the
required stage of growth, they should be removed from the
incubator and incubated on ice for 1.5 h prior to harvesting.
This results in a fourfold increase in transformation efficiency.
Longer incubations on ice result in reduced efficiency, probably
owing to increased cell lysis. This may also increase the possibility of arcing during the pulse delivery.
29. Competent cells that have been thawed from frozen should be
harvested and resuspended in fresh 10% glycerol prior to use to
remove any lysed bacteria. For M. smegmatis, we have found
that frozen cells are more competent than freshly prepared
cells.
30. It is important to have an even cell suspension for electroporation because any clumping of cells will lead to arcing and
282
Tanya Parish