0.5–500 ng DNA. In contrast, for M. avium, using increasing
amounts of DNA (up to 6 μg) does result in increased efficiency. The volume of DNA used is critical; for small volumes
of cell suspensions, the addition of a large amount of DNA in
water will alter the conductivity of the suspension. Therefore, it
is important that not more than 5 μL of DNA solution are
added to the cell suspension.
19. The use of 0.2-cm electrode gap cuvettes results in a higher
field strength than the use of 0.4-cm gap cuvettes.
20. Mycobacteria have chemically resistant cell walls that are difficult to lyse; thus, they are able to survive high voltage even
when pulses have long time constants. Several factors affect the
efficiency of transformation; these include the growth phase of
cells when harvested, electroporation media, and the field
strength and time constant of delivered pulse.
21. The use of a pulse-controller reduces the probability of arcing
when using high voltages applied to high-resistance media.
Factors that cause arcing include the presence of lysed cells in
the sample and salts in the DNA solution. Increasing the
parallel resistance to 1Ω increases the possibility of arcing;
therefore, a setting of 1000 Ω produces more consistent
results.
22. In some cases, arcing may be violent enough to blow the lid off
the electroporation cuvette, dispersing the cell suspension over
the inside of the electroporation chamber (thereby creating
aerosols). When working with pathogenic organisms, it is
imperative that the pulse is delivered with the electroporation
chamber placed inside a Class I safety cabinet and that appropriate disinfectants are at hand.
23. The dilution of cells immediately after the pulse is important.
Cells should be diluted at least 10-fold and incubated for
several hours prior to plating. Omission to this step leads to
greatly reduced efficiencies [24]. Presumably, the dilution
allows better recovery from the pulse and therefore greater
survival of transformants. Slow-growers should be incubated
for 3–16 h before plating; fast growers can be plated after
2–3 h.
24. The problem of clumping is also important when plating out
cells after electroporation. It is important to insure that resistant colonies have arisen from a single cell, so the cells must be
thoroughly resuspended before plating. Appropriate dilutions
may also help to alleviate this problem by thinning the resuspension. It is important to dilute the cells prior to plating
because the cell suspensions used for electroporation are very
concentrated. If the cells are not diluted, then it may be very
difficult to visualize true resistant colonies against a
Electroporation
281
amounts of DNA (up to 6 μg) does result in increased efficiency. The volume of DNA used is critical; for small volumes
of cell suspensions, the addition of a large amount of DNA in
water will alter the conductivity of the suspension. Therefore, it
is important that not more than 5 μL of DNA solution are
added to the cell suspension.
19. The use of 0.2-cm electrode gap cuvettes results in a higher
field strength than the use of 0.4-cm gap cuvettes.
20. Mycobacteria have chemically resistant cell walls that are difficult to lyse; thus, they are able to survive high voltage even
when pulses have long time constants. Several factors affect the
efficiency of transformation; these include the growth phase of
cells when harvested, electroporation media, and the field
strength and time constant of delivered pulse.
21. The use of a pulse-controller reduces the probability of arcing
when using high voltages applied to high-resistance media.
Factors that cause arcing include the presence of lysed cells in
the sample and salts in the DNA solution. Increasing the
parallel resistance to 1Ω increases the possibility of arcing;
therefore, a setting of 1000 Ω produces more consistent
results.
22. In some cases, arcing may be violent enough to blow the lid off
the electroporation cuvette, dispersing the cell suspension over
the inside of the electroporation chamber (thereby creating
aerosols). When working with pathogenic organisms, it is
imperative that the pulse is delivered with the electroporation
chamber placed inside a Class I safety cabinet and that appropriate disinfectants are at hand.
23. The dilution of cells immediately after the pulse is important.
Cells should be diluted at least 10-fold and incubated for
several hours prior to plating. Omission to this step leads to
greatly reduced efficiencies [24]. Presumably, the dilution
allows better recovery from the pulse and therefore greater
survival of transformants. Slow-growers should be incubated
for 3–16 h before plating; fast growers can be plated after
2–3 h.
24. The problem of clumping is also important when plating out
cells after electroporation. It is important to insure that resistant colonies have arisen from a single cell, so the cells must be
thoroughly resuspended before plating. Appropriate dilutions
may also help to alleviate this problem by thinning the resuspension. It is important to dilute the cells prior to plating
because the cell suspensions used for electroporation are very
concentrated. If the cells are not diluted, then it may be very
difficult to visualize true resistant colonies against a
Electroporation
281