MicroPulser Electroporator
MicroPulser
™
Electroporation Apparatus
Operating Instructions
and Applications Guide
Catalog Number
165-2100
For Technical Service Call Your Local Bio-Rad Office or in the U.S. Call 1-800-4BIORAD (1-800-424-6723)
Table of Contents
Page
Section 1 Safety Information .......................................................................................1
1.1 Electrical Hazards.......................................................................................................1
1.2 Mechanical Hazards ...................................................................................................1
1.3 Other Safety Precautions............................................................................................1
Section 2 Introduction..................................................................................................2
2.1 Overview of electroporation theory ...........................................................................2
2.2 Manipulation of instrument parameters.....................................................................4
Section 3 Factors Affecting Electroporation .............................................................5
3.1 Cell growth .................................................................................................................6
3.2 DNA............................................................................................................................6
3.3 Electroporation Media................................................................................................7
Section 4 MicroPulser Operating Instructions..........................................................9
4.1 Setting up the MicroPulser System............................................................................9
4.2 Operation of the MicroPulser.....................................................................................9
4.3 Electroporation using the MicroPulser ...................................................................11
Section 5 High Efficiency Electrotransformation of E. coli...................................12
5.1 Preparation of electrocompetent cells......................................................................12
5.2 Electroporation .........................................................................................................12
5.3 Solutions and reagents for electroporation ..............................................................13
Section 6 Electroporation of Staphylococcus aureus...............................................13
6.1 Preparation of electrocompetent cells......................................................................13
6.2 Electroporation .........................................................................................................14
6.3 Solutions and reagents for electroporation ..............................................................14
Section 7 Electroporation of Agrobacterium tumefaciens .....................................15
7.1 Preparation of electrocompetent cells......................................................................15
7.2 Electroporation .........................................................................................................15
7.3 Solutions and reagents for electroporation ..............................................................16
Section 8 Electroporation of Saccharomyces cerevisiae..........................................16
8.1 Preparation of electrocompetent cells......................................................................16
8.2 Electroporation .........................................................................................................16
8.3 Solutions and reagents for electroporation ..............................................................17
Section 9 Electroporation of Schizosaccharomyces pombe.....................................17
9.1 Preparation of electrocompetent cells......................................................................17
9.2 Electroporation .........................................................................................................18
9.3 Solutions and reagents for electroporation ..............................................................18
Section 10 Electroporation of Dictyostelium discoideum ..........................................18
10.1 Preparation of electrocompetent cells......................................................................18
10.2 Electroporation .........................................................................................................19
10.3 Solutions and reagents for electroporation ..............................................................19
Section 11 Electroporation of Pichia pastoris ............................................................19
11.1 Preparation of electrocompetent cells......................................................................19
11.2 Electroporation .........................................................................................................20
11.3 Solutions and reagents for electroporation ..............................................................20
Appendix I References .................................................................................................21
Appendix II Troubleshooting Guide for the MicroPulser ........................................22
Appendix III Product Information ................................................................................25
Warranty
Bio-Rad Laboratories warrants the MicroPulser against defects in materials and workmanship for
1 year. If any defects occur in the instrument during this warranty period, Bio-Rad Laboratories will,
at Bio-Rad's option, repair or replace the defective parts free of charge. The following defects, however,
are specifically excluded:
1. Defects caused by improper operation.
2. Repair or modification done by anyone other than Bio-Rad Laboratories or an authorized
agent.
3. Use of fittings or other spare parts supplied by anyone other than Bio-Rad Laboratories.
4. Damage caused by accident or misuse.
5. Damage caused by disaster.
6. Corrosion due to use of improper solvent or sample.
For any inquiry or request for repair service, contact Bio-Rad Laboratories after confirming the
model, serial number, invoice number, and purchase order number of your instrument.
Model
Catalog No.
Date of Delivery
Serial No.
Invoice No.
Purchase Order No.
Section 1
Safety Information
Read This Information Carefully Before Using The MicroPulser.
The MicroPulser meets the safety requirements of EN61010 and the EMC requirements
of EN61326 (for Class B, including flicker and harmonics).
1.1 Electrical Hazards
The MicroPulser produces voltages up to 3,000 volts and is capable of passing very high
currents. When charged to maximum voltage, the instrument stores about 50 joules. A certain
degree of respect is required for energy levels of this order. Safety system features prevent
operator access to the recessed input jacks and to the recessed electrode contacts inside the
sample chamber. These mechanical interlocks should never be circumvented.
There is high voltage present whenever the yellow pulse button is depressed and "PLS"
is shown in the light emitting diode display on the front of the instrument. If the capacitor
has been partially charged but not fired (for example, when the charging cycle has been
interrupted before the pulse is delivered), some charge may remain on the internal capacitor.
However, the user cannot make contact due to the system safety features.
1.2 Mechanical Hazards
The MicroPulser contains a patented arc-protection circuit that dramatically reduces the
incidence of arcing in the cuvette when high voltage is delivered into the sample. The unit
incorporates a circuit which senses the beginning of an arc and diverts current from the sample
within ~5 µsec, preventing, or greatly reducing mechanical, visual, and auditory phenomena
at the shocking chamber. Should an arc occur, the sample chamber is effective in containing
these small discharges, but nonetheless we strongly recommend wearing safety glasses when
using the instrument.
Do not use the MicroPulser with samples suspended in conductive media (refer to Section 3.3
for information on sample resistance).
1.3 Other Safety Precautions
Turn the unit off when not attended.
Avoid spilling any liquids onto the apparatus. Use only a paper towel or a cloth wet with
either water or alcohol to clean the outside surfaces of the MicroPulser.
Use only the Bio-Rad cables supplied with the MicroPulser.
Only use the shocking chamber in the assembled condition. Do not attempt to circumvent
the protection of the shocking chamber or use it while disassembled.
Verify the display segments periodically.
Do not use the MicroPulser if obvious case damage exists that exposes part of the inside
of the unit.
No user-serviceable parts are contained within the MicroPulser; the case should only be
opened by properly trained personnel.
1
2
Warning: The MicroPulser generates, uses, and radiates radio frequency energy. If it is not
used in accordance with the instructions given in this manual, it may cause interference with
radio communications. The MicroPulser has been tested and found to comply with the limits
for Class A computing devices (pursuant to Subpart J of Part 15 of FCC Rules) which provide
reasonable protection against such interference when operated in a commercial environment.
Operation of this equipment in a residential area is likely to cause interference. In this case the
user will be required, at their own expense, to take whatever measure may be required to correct
the interference.
Section 2
Introduction
2.1 Overview of Electroporation Theory
The MicroPulser system is used for the electroporation of bacteria, yeast, and other
microorganisms where a high voltage electrical pulse is applied to a sample suspended in a
small volume of high resistance media. The system consists of a pulse generator module, a
shocking chamber, and a cuvette with incorporated electrodes (Figure 1). The sample is placed
between the electrodes in the cuvette. The MicroPulser module contains a capacitor, which is
charged to a high voltage; the module then discharges the current in the capacitor into the
sample in the cuvette.
Fig. 1. MicroPulser consisting of Pulse Generator Module, Shocking Chamber and Cuvette.
The capacitance discharge circuit of the MicroPulser generates an electrical pulse with an
exponential decay waveform (Figure 2). When the capacitor is discharged into the sample, the
voltage across the electrodes rises rapidly to the peak voltage (also known as the initial voltage,
V0), and declines over time, t, as follows,
Vt= V0[e
-(t/τ)
] Equation 1
where τ = R x C, the time constant, a convenient expression of the pulse length. The resistance
of the circuit, R, is expressed in ohms, and the capacitance of the apparatus, C, is expressed
in microfarads. According to Equation 1, τ is the time over which the voltage declines to 1/e
(~37%) of the peak value. The internal circuitry of the MicroPulser is designed to provide
optimum electroporation of E. coli and S. cerevisiae, as well as many other microorganisms,
in which the optimum transformation efficiency occurs at a time constant of approximately
5 msec. These electroporation conditions are achieved by using a 10 microfarad capacitor
and by placing a 600 ohm resistor in parallel with the sample cuvette along with a 30 ohm
resistor in series with the sample cuvette.
Fig. 2. Exponential decay pulse from a capacitance discharge system. When the capacitor,
charged to an initial voltage, Vois discharged into cells, the voltage applied to the cells decreases over
time so that at time t = τ, the voltage is (1/e) x Voof the initial value.
In addition to the time constant, the electric field strength is the other instrument param-
eter that is important in determining transformation efficiency. The electric field strength, E,
is the voltage applied between the electrodes and is described by
E = V/d Equation 2
where V is the voltage applied and d is the distance (cm) between the electrodes. The strength
of the electric field and the size of the cells determine the voltage drop across each cell, and
it is this voltage drop that may be the important manifestation of the voltage effect in electroporation.
3
V
0
V
0
e
Time (msec)
Voltage (V)
The purpose of the 30 ohm series resistor in the MicroPulser is to protect the instrument
circuitry should arcing occur. Under normal operation, when samples are in high resistance
media, this resistor will not affect the voltage applied to the sample. However, this resistor will
significantly decrease the voltage applied to the sample if the resistance of the sample is low.
The fractional drop in voltage applied to the sample is given by
R30/ (R30+ R
sample
)
When R
sample
is 600 ohms, there is a 5% voltage drop to the sample [30 / (30 + 600) =
0.048]. For this reason, electroporation with the MicroPulser should not be performed in
solutions with a resistance of less than ~600 ohms. This includes samples in which the growth
medium was not adequately removed from the cells, DNA samples containing salt contributed
by residual sodium chloride, or ligation mixtures. The MicroPulser is able to measure the
resistance of the sample and will not pulse into very low resistance media.
2.2 Manipulation of Instrument Parameters
Several parameters on the MicroPulser may be altered to achieve maximum
transformation efficiency. These include the field strength, E, the time constant, τ, and the
width of a truncated exponential decay pulse. The field strength may be manipulated in two
ways. First, voltages between 200 and 3000 V may be set directly on the MicroPulser. This
parameter is the most easily controlled. The process of varying the voltage while keeping all
other conditions unchanged is the basis for most electroporation optimization procedures.
Second, using cuvettes with different electrode gap widths permits a means of changing the
field strength. For electroporation of microorganisms, 0.1 and 0.2 cm gap cuvettes are most
often used. Electroporation of E. coli is generally carried out at a voltage of 1.8 kV
(E = 18 kV/cm) when electroporating cells in 0.1 cm cuvettes and at a voltage of 2.5 kV
(E = 12.5 kV/cm) when electroporating cells in 0.2 cm cuvettes. These electroporation
conditions are pre-programmed into the MicroPulser as programs Ec1 (V = 1.8 kV) and Ec2
(V = 2.5 kV) in the bacterial settings menu. In addition, a third program, Ec3 in the bacterial
settings menu, delivers a voltage of 3.0 kV (E = 15 kV/cm in 0.2 cm cuvettes) which we have
found results in even higher transformation efficiency compared to electroporation at 2.5 kV.
The time constant may be altered by changing the sample resistance. The sample
resistance may be manipulated in two ways. First, increasing the salt or buffer concentration
of the electroporation media decreases the resistance of the sample, and vice versa, resulting
in a change in the time constant. Second, the volume of the sample in the cuvette is inversely
proportional to the resistance of the sample; decreasing the sample volume increases the
sample resistance. This effect of volume on sample resistance is most noticeable in low
resistance media. These effects are discussed further in Section 3.3.
4
The MicroPulser also includes a means to truncate the exponential decay pulse sooner
than the expected time constant as long as the voltage is greater than 600 V. When the pulse
is terminated by the MicroPulser, voltage is applied to the sample only for the specified time,
which may be between 1.0 and 4.0 msec. Figure 3 shows how this waveform differs from
the true exponential decay pulse.
Fig. 3. Truncation of an exponential decay pulse by the MicroPulser. The solid line shows the voltage applied to the cells as a function of time during a pulse terminated after 2.5 msec. The dashed line
shows the voltage that would normally be applied to the cells during a true exponential decay pulse.
Section 3
Factors Affecting Electroporation
The electrical conditions for the electroporation of microorganisms have been verified
through years of research (see Chang, et al., 1992, and Nickoloff, 1995, for overviews as well
as for protocols on electroporation of numerous species). For many microorganisms, optimum electrotransformation occurs under electrical conditions relatively similar to those used
for E. coli and S. cerevisiae, two species that are most commonly used in research today. For
electroporation of E. coli, conditions reported as being used most often are 0.2 cm cuvettes containing 40 µl of cells at a voltage of 2.5 kV and a time constant of ~5 msec. For
electroporation of S. cerevisiae, conditions reported as being used most often are 0.2 cm
cuvettes containing 40 µl of cells at a voltage of 1.5 kV and a time constant of ~5 msec. For
many bacterial species, including Salmonella, Pseudomonas, Helicobacter, Borrelia,
Streptococcus, Lactococcus, and Enterococcus, the conditions for electroporation are
identical to those used for E. coli. For many other bacterial species, altering the field strength
will often result in higher electrotransformation. A similar case is found with other species of
yeast.
The MicroPulser is designed to deliver precisely those pulse parameters needed for the
highest transformation efficiency of E. coli and S. cerevisiae. The time constant has been set
5
Voltage (V)
V
0
V
0
e
2.5
5
Time (msec)
at 5 milliseconds when working with high-resistance samples. For these organisms, the
MicroPulser has pre-programmed settings for delivery of the correct voltage when
electroporating E. coli in either 0.1 or 0.2 cm cuvettes, or when electroporating S. cerevisiae
in either 0.2 or 0.4 cm cuvettes.
3.1 Cell Growth
For most bacterial species, the highest transformation efficiencies are obtained when cells
are harvested in early to mid-log growth. For E.coli, as the cells reach stationary phase, the
transformation efficiency will decline precipitously (Dower, 1990). In contrast, most yeast
species are generally harvested in mid- to late-log growth. For S. cerevisiae, the
transformation efficiency increases as much as 60-fold from early to late-log cultures (Becker
and Guarente, 1991). The optimal portion of the growth phase to harvest cells is generally
dependent on the cell type. When preparing competent cells of a new species it is generally
best to employ conditions worked out for use with the same genus. Suggestions for factors to
consider and general methods for producing electrocompetent cells are discussed in the
articles by Dower et al. (1992) and Trevors et al. (1992).
3.2 DNA
While the majority of electroporation applications involve delivery of plasmid DNA to
cells, it should be mentioned that nearly any type of molecule can be introduced into cells by
electroporation, including RNA, proteins, carbohydrates, and small molecules. With few
exceptions, when delivering autonomously replicating plasmids, the highest transformation
efficiencies are obtained when electroporating supercoiled plasmid. However, electroporating
plasmid that will integrate into the host genome is usually most efficient using linear
plasmid. For example, Candida, Pichia, and Tetrahymena are transformed more efficently
when transformed with linearized than with supercoiled integrating plasmids.
In both E. coli and Listeria monocytogenes, the transformation efficiency of relaxed
circular plasmid is only slightly lower than that of supercoiled plasmid (Leonardo and Sedivy,
1990, Park and Stewart, 1990). However, linear plasmid is about 103- 104-fold less efficient
than the corresponding circular plasmid in both E. coli and Streptococcus pyogenes
(Shigekawa and Dower, 1988, Simon and Ferretti, 1991). Electroporation efficiency per mole
of plasmid generally decreases as the plasmid size increases in numerous species, including
E. coli (Leonardo and Sedivy, 1990, Siguret et al., 1994), Pseudomonas aeruginosa (Dennis
and Sokol, 1995), and Streptococcus thermophilus (Somkuti and Steinberg, 1988). However,
in some species, including Lactococcus lactis (Holo and Nes, 1995), Enterococcus faecalis
(Cruz-Rodz and Gilmore, 1990), and Clostridium perfringens (Allen and Blaschek, 1990),
transformation efficiency appears to be independent of plasmid size up to 20–30 kb.
Although transformation of most microorganisms has been accomplished using plasmid
DNA isolated by a variety of methods, the plasmid purity has an effect on transformation
efficiency. Significantly lower transformation efficiencies are generated with unpurified
miniprep plasmid DNA than with plasmid DNA purified by a variety of procedures. Plasmid
produced using the Bio-Rad Quantum matrix is as efficient as CsCl-purified plasmid for
transformation of microorganisms.
Generally, for all types of microorganisms, the frequency of transformation increases
with inceasing DNA concentration in the electroporation buffer. For E. coli, the frequency of
transformation (transformants/survivor) is dependent on DNA concentration over at least six
orders of magnitude (10 pg/ml to 7.5 µg/ml); within this range the DNA concentration
determines the probablility that a cell will be transformed. At the higher DNA concentrations,
up to 80% of the survivors are transformed (Dower et al., 1988). Because the number of
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