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ELECTROPORATION
OPTIMISATION GUIDE
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CHAPTER I: INTRODUCTION 7
CHAPTER II: ELECTROPORATION-MOST IMPORTANT PARAMETERS 3
Electrical Factors 3
Other Parameters 3
CHAPTER III: BACTERIAL ELECTROPORATION 5
Introduction 5
How to Prepare Bacteria for Electroporation 5
Electroporation of E.Coli 6
Output Voltage and Electric Field Using 1&2mm Cuvettes 7
Optimization Advice for Gram-Bacteria 7
Optimization Advice for Gram+Bacteria 7
General Optimization Advice for Bacteria 8
Troubleshooting ~ Bacterial Electroporation 8
CHAPTER IV: MAMMALIAN CELL ELECTROPORATION 11
Introduction 11
Cell Harvesting 11
Standard Cell Line Electroporation 12
Optimization Advice for Mammalian Cell Lines 13
Optimization Advice for Primary Mammalian Cells 13
General Optimization Advice for Mammalian Cells 14
Troubleshooting ~ Mammalian Cell Electroporation 15
CHAPTER V: YEAST ELECTROPORATION 19
Yeast Preparation for Electroporation 17
Electroporation 17
Optimization Advice for Yeast 18
General Optimization Advice for Yeast 18
Troubleshooting ~ Yeast Electroporation 19
CHAPTER VI: SPECIAL APPLICATIONS 21
Fish, birds and other eukaryotic cell electroporation 21
Standard parameters for using the optipulse option 21
Tissue electroporation using 10mm cuvettes 22
CHAPTER VII: INTACT PLANT ELECTROPORATION 23
CHAPTER I: INTRODUCTION
The actual mechanism is still not fully understood, other than the thermal
motion for small molecules. For the macromolecule transport, it is known that
the polarity is important and involved in the electrophoretic movement of the
DNA v,vi into the cell.
OPTIMISATION GUIDE
Electrical Factors
Cell permeability induced by electroporation is dependent on the following
parameters:
The electric field (E) and pulse duration (τ) depends on the reciprocal of cell
diameter (see chapter 13 for electrical background). The electric field to get
efficient electroporation varies from 450V.cm-1 for large insect cell to 12.5
kV.cm-1 and higher for bacteria.
The electric field and pulse duration has to be above the lower working limitvii.
Pore number and pore diameter increase with the product of the electric field
and pulse duration. However, if the upper working limit is reached this causes
the cell to be irreversibly damaged.
Other Parameters
Results will also depend on the following parameters:
The purity of the molecule to be transferred. Impurities that will be transferred
into the cells during electroporation have potential unpredictable side effects,
and generally will increase cell mortality. In chapter 10 we describe
techniques for DNA purification.
The purity of water and other chemicals in contact with the cells during the
electroporation process is also critical for the same reasons described here as
above. Freshly prepared 18~MOhms water (MilliQ®) should be used
whenever it is possible.
The chemicals used for the preparation of the samples should be of molecular
biology grade and usage should be restricted to electroporation only.
The cleanness of the vessel in contact with sample is also very important.
Detergents are known to dramatically decrease transformation efficiency.
Disposable vessels should be used wherever it is possible. In any case,
vessels contaminated with detergents should be avoided for the preparation of
the samples and to store these chemicals, including water.
1
Some cuvette manufacturers suggest this type of sterilisation. We strongly recommend not to use this method!
Cell preparation and growth phase. Cell preparation is very important. Fast
growing cells are usually considered better for electroporation2.
2
However it is now possible to electroporate efficiently quiescent cells with the InSitu electroporation system from
Thermo
OPTIMISATION GUIDE
Introduction
The conditions for electroporation efficiency vary between different cell types.
However, for most of the bacteria the optimum electric field is 12,5kV.cm-1 or
18,0kV.cm-1, and the optimum pulse time is 5ms. Usually you will use an
output voltage of 2.5kV with the 2mm cuvettes and 1.8kV with the 1mm
cuvettes.
Water quality is vital; we recommend you only use freshly prepared 18MΩ,
stored in detergent free clean vessels, or ideally in a disposable vessel.
3
Never store this type of water in vessels, which have been washed with detergent. Disposable vessels are strongly
recommended for best result.
Electroporation of E.Coli
1. Defrost an aliquot of electrocompetent cells.
2. Load an Eppendorf tube chilled on ice with 40µl4 of cell
suspension.
3. Add 1 to 5µl of ligation mix (DNA).
4. Mix well and keep on ice for 1 minute.
5. Select 2500 Volt as the output voltage.
6. Load a 2mm cuvette chilled on ice with the cell suspension.
Avoid putting your finger on the aluminium electrodes or it will
dramatically increase the temperature of the sample and increase
the risk of arcing.
7. Trigger the pulse immediately.
Cuvette 2mm
Voltage 2500 Volts
Capacitor 25µF5
Shunt Resistor 201R
4
If you are using Thermo electroporation cuvettes you will be able to reduce the volume to 20µl with the 2mm
model (Cat.# ECU-102) and to 10µl with the 1mm model (Cat.# ECU-101). These extremely low volumes are
possible because of the special V-shape design of Thermo cuvette. If you are using commercial electrocompetent
cells you will achieve significant economy.
5
25µF and 200R is the most common combination of capacitor and resistor to get the 5ms optimal pulse time.
Some equipment are offering other combination resulting in 5ms pulse (EasyjecT Prima and Optima 15µF and
335R, Biorad MicroPulser® 10µF and 500R, EC-630 from BTX 40µF and +/- 130R
6
Typical 5ms pulse time duration are obtained selecting a 25µF capacitor and a shunt resistor of 200Ω
7
Typical 5ms pulse time duration are obtained selecting a 25µF capacitor and a shunt resistor of 200Ω
Low efficiency Transfer time too long After the pulse the cells must be
transferred immediately (max. 30s) in a
good quality outgrowth medium.
Introduction
The conditions for electroporation efficiency vary between cell types.
However, for most of the mammalian cells lines 8 the optimum electric field is
650 V.cm-1 with a pulse time of a few tens of milli-secondsviii. Usually you will
use an output voltage of 250 V and a capacitor value of 1500µF with the 4mm
cuvette loaded with 800µl of cell suspension.
Cell Harvesting
It is highly recommended to use the normal harvesting procedure. Any change
could affect the cell capability to survive the electroporation treatment.
8
Primary cells usually require higher electric field i.e. 750.cm-1
9
You can prepare the sample into the cuvette but is more difficult to mix.
10
See DNA purification section in this book, Chapter X.
Fast growing cells usually give better results. For cells growing adherent11,
we recommend to harvest at 50% to 70% confluence.
All chemicals used should be dedicated to cell culture and preparation and
should be a molecular biology grade.
Cuvette 4mm
Voltage 250 Volts
Capacitor 1500µF
Shunt Resistor …… (Infinite)
11
However it is now possible to electroporate efficiently quiescent cells with the InSitu electroporation system
from Thermo
After the electrical pulse it is critical to transfer the cells in fresh medium within
seconds. During the pulse electrolyses will dramatically change the buffer
composition. The medium will change in a very hostile environment for the
treated cells and could result in a dramatic decrease of the experiment
efficiency. Never incubate cells in the cuvette after the pulse.
Below you will find the “window” of the most usual voltage output use for the
electroporation of mammalian cell lines with 4mm cuvette and a sample
volume of 800µl.
Bear in mind that other parameters like the DNA quality, the electroporation
medium and the transfer time are also extremely important for the success of
an electroporation experiment.
12
See Thermo Hybaid newsletter October 1997 for more details.
Keep in mind those other parameters like the DNA quality, the electroporation
medium and the transfer time are also extremely important for the success of
an electroporation experiment.
Check the killing rate under standard. If you kill less than 50% of the cells,
optimise the voltage by 10 Volt increments. Check “wi ndow” of figure 03 and
04 for efficient output voltage.
If you kill more than 50% of the cells and less then 90%, optimise the voltage
by 10 Volt steps around the standard value.
If you kill more than 90% of the cells, decrease the voltage by 10~Volts steps.
Further Optimisation is possible through the capacitor value, which will
influence the pulse time. To proceed, vary the capacitor value by 150µF
increments up and down the initial standard value.
Low efficiency Too high electric field Decrease the output voltage by 10-
and high death volts increments.
rate.
It is considered normal to kill a
minimum of 50% of the cells and up to
90%.
Impure DNA DNA should be purified as described
in Annex A or with other methods
giving similar purity grade.
CsCl purification alone or fast
purification techniques are not always
reliable.
Check the promoter efficiency in the
transformed cell line
Low efficiency Too low voltage apply Increase the output voltage by 10-
and low death volts step.
rate.
It is considered normal to kill a
minimum of 50% of the cells.
No signal at all Non-expressible Make sure that the cell line can
sequence, weak express DNA of interest and that it
promoter or cells unable has a strong promoter to drive its
to express gene expression. Use a positive control.
The protocol has been optimised for Saccharomyces cerevisae is valid for
most yeast strains. However, growth conditions, growing medium, outgrowth
medium and selection methods will need to be adapted.
Electroporation
1. In a cold 1.5ml Eppendorf tube mix together the DNA (optimum
amount of DNA is 100ng) and 40µl of yeast suspension. Keep on
ice for 3 minutes.
2. Transfer the DNA/Yeast mixture in a cold 2mm-electroporation
cuvette. The quality of the DNA influences strongly the
transformation frequency. Incubate on ice for 5 minutes.
Cuvette 2mm
Voltage 1700 Volts
Capacitor 25µF
Shunt Resistor 486R (or closest
value)
486R
335R
1400 1500 1600 1700 1800V
Output Voltage
Figure 05: Efficient output voltage and shunt resistor “window” for the
electroporation of yeast.
Chemicals grade. All chemicals used should be dedicated to cells culture and
preparation and should be of molecular biology grade.
Low efficiency Transfer time too long After the pulse the cells must be
transferred immediately (max. 30s) in
a good quality outgrowth medium.
Cuvette quality For best results we recommend using
the highest quality disposable cuvette.
It is not recommended to reuse
electroporation cuvettes. During the
electroporation an aluminium oxide
deposit will form on the electrodes.
The thickness of this highly resistive
OPTIMISATION GUIDE
Cuvette 4mm
Voltage 250 Volts
Capacitor 1500µF
Shunt Resistor …… (Infinite)
Temperature Room Temp.
Cuvette 4mm
Sample Volume* 800µl
Electroporation OptiBuffer (# EPEKITE1) or Usual growth medium
Medium OptiBuffer should be tested in this type of application
Electroporation
Room temperature
Temperature
Cell concentration 5 x 106 cells / ml
Pre incubation Before the pulse incubate DNA with the cells for minimum 1
time** min to maximum 5 min.
30µg – Purity of DNA is critical (See the EquiBio
DNA quantity*** electroporation Optimisation guide for recommended
methods of purification)
OptiPulse Electroporation
Electroporation Parameters
Cuvette 10mm
Sample Volume* 1000µl
Electroporation Usual growth medium or OptiBuffer (# EKIT-E1)
Medium
Electroporation
Room temperature
Temperature
Cell concentration N/A
Before the pulse incubate DNA with the tissue for
Pre incubation time**
minimum 1 min to maximum 5 min.
DNA quantity*** 30µg
OPTIMISATION GUIDE
Plant Preparation
This method as been published in Molecular Biotechnology (1995), Volume 3,
17-23, G.M. Chowrira, V. Akella, and P.F. Lurquin, Electroporation-Mediated
Gene Transfer into Intact Nodal Meristems In Planta. Optimised for pea (var.
Sparkle), lentil (var. Crimson), soybean (var. Wye), and cowpea (var.
Blackeye 5). Personal communication (1997) from A.MC Collén I. De Jong
and C. I. Jarl Transformation of the legume Galegae orientalis L.
13
Thermo commercialises 2 polycationic lipids: EasyFector cat. # ETR-E1 and PrimeFector cat. # ETR-P1.
Cuvette 10mm
Voltage 300 Volts
Capacitor 900µF
Shunt Resistor …… (Infinite)
Temperature Room Temp.
3. Remove the bud from the cuvette as soon as possible after the
pulse.
4. The electroporated buds on the plant are allowed to grow, bear
flowers, and set seeds. Control plants are electroporated in the
same manner as described above, but with pUC8 vector DNA.
5. Leaf samples for transient expression of the gene are collected 3
to 4 weeks after electroporation.
6. Stable transformation is confirmed in the leaf samples of plants
originating from the seeds of the electroporated plants, by
Southern/Northern/Western blot analysis.
Note: The above protocol can be used both to study transient
expression of transgenes in organs issued from the
electroporated bud and to produce transgenic plants.
Injection of DNA i nto the nodal meristem prior to electroporation is crucial for
increased transformation frequencies, but care must be taken so as not to
damage the meristem during the operation.
OPTIMISATION GUIDE
Cuvette 4mm
Voltage 280 Volts
Capacitor 1050µF
Shunt Resistor 201R
Temperature Room Temp.
14
0.2M to 0.6M depending on the cell type.
OPTIMISATION GUIDE
Cuvette 4mm
Voltage 230Volts
Capacitor 900µF
Shunt Resistor …… (Infinite)
Temperature Room Temp.
Introduction
The quality of the DNA STRONGLY influences the results of transfection
experiments. Therefore only DNA of the highest quality, which is completely
free of contaminating RNA, ribo-nucleotides, genomic DNA or proteins, should
be used in transfection experiments. Impurities in DNA including ribo-
nucleotides and endoto xins released during affinity chromatography
purification increase cell mortality in electroporation e xperiments. There are a
number of endotoxin free purification kits on the market (Qiagen® &
Stratagene®), which may be a suitable alternative to the CsCl method.
Electroporation carried out using purified DNA as described here below will
induce a linear increasing of efficiency with the DNA concentration used.
Purification of Closed Circular DNA
1. Measure the volume of the DNA solution. For every millilitre, add
exactly 1g of solid CsCl. Warm the solution to 30°C to facilitate
the dissolution of the salt. Mix the solution gently until the salt is
dissolved.
2. Add 0.8ml of a solution of ethidium bromide (10mg.ml-1 in water)
for every 10ml of the DNA~CsCl solution. Immediately mix the
ethidium bromide solution (which floats on the surface) with the
denser DNA~CsCl solution. The final density of the solution
should be 1.55g/ml (refractive index = 1.3860), and the
concentration of ethidium bromide should be approximately
740µg.ml-1.
3. Stock solutions of ethidium bromide should be stored in light-tight
containers (e.g., in a bottle completely wrapped in aluminium foil)
at room temperature. Caution: Ethidium bromide is a powerful
mutagen and is moderately toxic. Gloves should be worn when
working with solutions that contain this dye. After use, these
solutions should be decontaminated.
4. Centrifuge the solution at 8000 rpm for about 5 minutes at room
temperature in Sorvall SS34 rotor (or its equivalent). The furry
scum that floats to the top consists of complexes formed between
the ethidium bromide and bacterial proteins.
5. Using a Pasteur pipette or a disposable syringe fitted with a large-
gauge needle, transfer the clear, red solution under the scum to a
tube (Beckman Quick-Seal or equivalent) suitable for
centrifugation in a Beckman vertical Ti65 rotor or an angle Ti50,
DoublePulse Electroporation
DoublePulse methodologies were first used in the late 1980’s with the launch
of commercially available electroporators xi. Improved results were achieved
by generating a relatively high electric field with a short duration for the first
pulse, followed 0.1 seconds later by a long pulse with a low electric field. The
first pulse is thought to optimise the pore size and number, the second is
thought to move the DNA into the cell in a fashion similar to electrophoresis.
Both pulses together have a reduced energy level (Figure 2), thus increasing
cell survival.
OptiPulse™
The DoublePulse method has been proven to produce improved results over
single pulse methodologies and is regularly used on difficult cells. At Thermo
we have realised that while DoublePulse offers some advantages over single
pulse, a new discharge method “OptiPulse“ is available on Thermo’s CelljecT
Pro and can be used in addition or as a replacement to single or
DoublePulse methodologies. The OptiPulse is essentially a tuned
DoublePulse discharge, with the second pulse being released before the first
discharge is completely dissipated.
Shake until the solutes have dissolved. Adjust the pH to 7.0 with 5N NaOH
(~0.2ml). Adjust the volume of the solution to 1 litre with de-ionised H2 O.
Sterilise by autocla ving for 20 minutes at 15lb/sq. in. on liquid cycle.
SOB Medium
Per litre: To 950ml of de-ionised H2O, add:
Bacto-tryptone 20g
Bacto-yeast extract 5g
NaCl 0.5g
Shake until the solutes have dissolved. Add 10ml of a 250mM solution of KCl.
(This solution is made by dissolving 1.86g of KCl in 100ml of de-ionised H2 O.)
Adjust the pH to 7.0 with 5N NaOH (~ 0.2ml). Adjust the volume of the
solution to 1 litre with de-ionised H2O. Sterilise by autoclaving for 20 minutes
at 15lb/sq. in. on liquid cycle.
Just before use, add 5ml of a sterile solution of 2M MgCl2 . (This solution is
made by dissolving 19g of MgCl2 in 90ml of de-ionised H2O and sterilise by
autoclaving for 20 minutes at 15lb/sq. in. on liquid cycle.)
SOC Medium
SOC Medium is identical to SOB medium, except that it contains 20mM
glucose. After the SOC Medium has been autoclaved, allow it to cool to 60°C
or less and then add 20ml of a sterile 1M solution of glucose (this solution is
made by dissolving 18g of glucose in 90ml with de-ionised H2O). After the
pH HCl
7.4 70ml
7.6 60ml
8.0 42ml
pH 7.6
19mM Tris · Cl (pH 7.6)
1mM EDITA 9pH 8.0)
pH 8.0
10mM Tris · Cl (pH 8.0)
1mM EDTA (pH 8.0)
STE (Also Called TEN)
0.1M NaCl
10mM Tris · Cl (pH 8.0)
0mM EDTA (pH 8.0)
Electric Field
The electric field 15, E, is expressed in Volts per centimetre. It can be
calculated with the following formula:
E = V.d -1
15
For the Gram-negative bacteria E.Coli the electric field necessary to achieve good transformation efficiency is
12,5kV * cm -1. To achieve this electric field it will be necessary to set up the output voltage of the electroporation
system at 2500V with 2mm cuvette (2500 * 0.2-1) or to 1250V with 1mm cuvette (1250 * 0.1-1).
P = 0.5.C.V 2
The energy is expressed in Joule (J) while the capacitor is expressed in Farad
and the voltage in Volts. The energy will vary dramatically with the voltage
while being less influenced by the capacitor value. This explains why it is
necessary to use a shunt resistor in high voltage 16 electroporation to avoid
destruction of the sample.
Pulse Time
By definition the pulse time (τ) is the elapsed time, in seconds, from the
beginning of the pulse, when the electric field is maximum (E0), until the
electric field has decreased to e-1 (0.368) of the initial value E 0. Practically this
value is measured by the microprocessor of the electroporation unit.
16
In case of E.Coli electroporation, the recommended capacitor value is 25µF and the output voltage is 2500 V.
The resulting energy stored in the capacitor is very high 156.25 J [(25 x 10-6) x (25002)].
The pulse time can be calculated for an ideal system using the following
formula:
τ = R.C
Pulse time 17 (τ) is expressed in seconds (s) when the resistance value is
expressed in Ohms (Ω or R) and the capacitance value in Farad.
F IGURE 0 8 :
E LECTROPORATION S YSTEM; B ASIC DESIGN FOR LOW
VOLTAGE ELECTROPORATION. N O SHUNT RESISTOR IS NECESSARY .
This is the design used for low voltage electroporation, typically for
mammalian cells, and in general for eukaryotic cells. In this type of experiment
the capacitor (C) is completely discharged through the sample (RLoad).
For this type of experiment the sample (RLoad) usually has a relatively low
resistance value (1kΩ to 10kΩ) due to the electroporation buffer used (PBS,
HBS, culture medium or OptiBuffer used at room temperature).
The pulse time will obey the simple pulse time formula (τ = R.C) with R =
RLoad. However it is impossible to calculate the pulse time, same if RLoad is
known, because the sample resistance will decrease dramatically during the
pulse. Usually the measured pulse time is about a tenth of the computed
value.
17
In case of E.Coli electroporation, the recommended capacitor value is 25µF and the recommended shunt resistor
value is 200 R the resulting calculated pulse time is 5 milliseconds [(25 x 10-6) x 200] = 0.005s.In case of bacteria
electroporation the calculated value and the measured value are very close.
A typical value for E.Coli electroporation are 200Ω for Rs and 100 000Ω for
RL. This means that the resulting resistance of the circuit will be 199.6Ω (R-
1
=200-1+100000-1). This example shows that the shunt resistor will absorb
nearly all the energy of the pulse thus protecting the cells and will regulate the
pulse time. It also demonstrates that the resistance of the sample must be as
high as possible. Usually electroporation at high voltage of a low resistance
sample will result in arcing and the loss of the sample.
For this type of experiment the sample (RL) usually has a very high resistance
value (> 50kΩ) due to the electroporation buffer used (usually 18 MΩ water at
ice temperature).
Electroporation Equipment
EP EJ 002 Cellject PRO
Lipofection Products
i
Kinosita, K. Jr., and Tsong, T. Y. (1977). Hemolysis of human erythrocytes by a transient electric field. Proc. Natl. Acad. Sci.
USA 74, 1923-1927.
ii
Neumann E.A., Sowers E.and Jordan C. (1989), Electroporation and Electrofusion in Cell Biology. Plenum Press, New York,
436.
iii
Zimmermann U., Vienken, J., and Pilwat, G. (1980). Development of drug carrier systems: electric field induced effects in cell
membranes. J. Electroanal. Chem. 116, 553-574.
iv
Potter H. (1992), Protocols for using electroporation to stably or transiently transfect mammalian cells. In Guide for the
electroporation and electrofusion. Chang D.C., Chassy B.M., Saunders J.A., and So wers A.E. Academic Press, San Diego, 457-
464.
vi
Tien-An Yang, William C. Heiser and John M. Sedivy (1995), Efficient InSitu electroporation of mammalian cells grown on
microporous membranes. Nucleic Acids Research, 23 (15), 2803-2810.
vii
Hendrick Wolf, Marie Pierre Rols, Elvira Boldt, Eberhard Neumann and Justin Teissié (1994), Control by Pulse Parameters of
Electric Field-Mediated gene Transfer in mammalian cells. Biophysical Journal, 66, 524-531.
viii
Christopher Baum, Peter Forster, Susanna Hegewisch-Becker and Klaus Harbers (1994), An Optimized Electroporation
Protocol Applicable to a Wide Range of Cell Lines. BioTechniques, 17 (6), 1058-1062.
ix
J.M. Teare, R. Islam, R. Flanagan, S. Gallagher, M.G. Davies and C. Grabau (June 1997) Biotechniques 22 (6), 1170-1174.
x
William W. Wilfinger, Karol Mackey and Piotr Chomczynski (March 1997), BioTechniques, 22 (3), 474-481.
xi
The CelljecT from Thermo Hybaid, launched 1990, was replaced by the CelljecT Pro in 1992.