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THERMO ELECTRON CORPORATION

ELECTROPORATION

OPTIMISATION GUIDE

Thermo Electron Corporation


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© Thermo Electron Corporation 2003 Issue 4.0, June 2003 ii


THERMO ELECTRON
ELECTROPORATION OPTIMISATION GUIDE
TABLE OF CONTENTS

Page
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

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 iii


Plant Preparation 23
Plant Bud Electroporation 24
General Optimization Advice for Intact Plant 24
CHAPTER VIII: PLANT PROTOPLAST ELECTROPORATION 27
Plant Protoplast Preparation 27
Plant Protoplast Electroporation 27
General Optimization Advice For Plant Protoplast 27
CHAPTER IX: PROTEIN, OLIGO AND SMALL MOLECULE ELECTROPORATION29
CHAPTER X: DNA PURIFICATION 31
Introduction 31
Purification of Closed Circular DNA 31
Removal of RNA From Preparation of Plasmid DNA 32
Desalting of Ligation Mix 33
CHAPTER XI: DOUBLE & OPTIPULSE 35
DoublePulse® Electroporation 35
Energy Level Single Pulse versus DoublePulse 35
OptiPulse™ 36
CHAPTER XII: MEDIUM AND BUFFERS 38
LB Medium (Luria-Bertani Medium) 36
SOB Medium 36
SOC Medium 36
2x HEPES-Buffered Saline 37
Phosphate-Buffered Saline (PBS) 37
1M Tris 37
TE 37
STE (Also Called TEN) 37
STET 38
TNT 38
CHAPTER XIII: GENERAL INFORMATION 40
The Cleanliness of the Glassware and Plasticware. 40
Transfection Principle 40
Transient Transfection 40
Stable Transfection 40
Primary Cells and Cell Lines 40
Primary Cell Culture 40
Finite Cell Line 41
Established Cell Line 41
Adherent and Suspension Cells 41
Adherent Cells 41
Suspension Cells 41
Media and Supplements 41
Serum 41
Transfection Methods 41
Plasmid DNA Quality 42
Conformation and Concentration of DNA 42
Genetic Reporter Systems 42
Commonly Used Reporter Genes 42

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 iv


Chloramphenicol Acetyltransferase 42
Firefly Luciferase 42
B-Galactosidase 43
Human Growth Hormone (HGH) 43
Green Fluorescent Protein 43

CHAPTER XIV: ELECTRICAL BASICS 43


Electric Field 43
Capacitor 44
Pulse Energy 44
Pulse Time 44
Basic Designs for Low Voltage Application 45
Total Resistance and Pulse Time 46
Basic Design for High Voltage Application 46
CHAPTER XV: ORDERING INFORMATION 40

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 v


© Thermo Electron Corporation 2003 Issue 4.0, June 2003 vi
THERMO ELECTRON ELECTROPORATION
OPTIMISATION GUIDE

CHAPTER I: INTRODUCTION

During electroporation a trans-membrane electric field is induced by an


external electric field, usually a short Direct Current (DC) pulse being applied
to the cell suspension. This trans-membrane electric field induces local
destabilisationi of the cell membrane, commonly called electropores ii.

During the destabilisation period, the membrane is highly permeable to small


molecules iii like ions a nd water, but also to macromolecules as large as DNA iv .
If suitable electrical pulse parameters are used, the cells will recover, the
electropores reseal spontaneously and continued growth is ensured.

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.

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 vii


© Thermo Electron Corporation 2003 Issue 4.0, June 2003 2
THERMO ELECTRON ELECTROPORATION

OPTIMISATION GUIDE

CHAPTER II: ELECTROPORATION-MOST IMPORTANT


PARAMETERS

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.

The quality of the electroporation cuvette used is extremely important. Top


quality cuvettes should be used for critical experiments. Reusing the cuvette is
common but the transformation efficiency will decrease over a period of time
because the oxidation of the aluminium will change the electroporation
conditions by adding an extra serial resistance into the circuit. Steam 1
sterilisation speed-up the oxidation process and should be avoided. Reusing

1
Some cuvette manufacturers suggest this type of sterilisation. We strongly recommend not to use this method!

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 3


the cuvette increases the risk of contamination and more seriously increases
the risk to transfer a wrong DNA.

Post electroporation events. Due to the electrolysis of the electroporation


buffer, after the pulse the cells are in a very hostile environment and must be
diluted and transferred as quickly as possible (less than 30 seconds) in the
appropriate outgrowth medium.

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

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 4


THERMO ELECTRON ELECTROPORATION

OPTIMISATION GUIDE

CHAPTER III: BACTERIAL ELECTROPORATION

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.

How to Prepare Bacteria for Electroporation


This is an optimised protocol for E.Coli. Please adapt it according to the
bacteria strain used.

1. Prepare a 10ml pre-culture on LB medium. For best results, avoid


using over-night pre-culture.
2. Dilute pre-culture as follows: 4 ml in 200-ml of fresh LB pre-
warmed at 37°C.
3. Grow the cells at 37°C.
4. When OD(600)=0.6 is reached, chill the culture on ice as quickly
as possible.
5. Centrifuge in disposable tubes (50ml disposable type) for 5
minutes at 3000 rpm.
6. Re-suspend the pellets in 25ml freshly prepared water 3 (MilliQ®
quality) at ice temp.
7. Repeat steps 5 & 6 twice more.
8. Re-suspend the pooled pellets in 400µl (cell concentration should
be 1 x 1010 cells x ml-1) freshly prepared water (MilliQ® quality) at
ice temp.
9. Check the final volume and add 10% of glycerol (molecular
biology grade).
10. Use immediately or aliquot the electrocompetent cells to 100µl in
10% glycerol and freeze at minus 70°.

The following advice is valid for any kind of bacteria cells:


Fast growing cells usually give better results
Temperature decrease after stopping the cell growth must be as fast as
possible.
Carry out all post growing steps rapidly.

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.

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 5


All chemicals used should be dedicated to electrocompetent cell preparation
and of a molecular biology g rade.

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

8. As soon as possible (less than 30 seconds) re-suspend the cells


in the cuvette with 1ml SOC medium (the quality of the SOC is
important).
9. Transfer the cells in an appropriate vessel and incubate at 37°C
for 1 hour.
10. Plate the cells on the selective medium.
11. After approximately 24 hours read the results.

The following advice is valid for all bacteria cells:


Prior to electroporation samples and cuvettes must be stored on ice.
Transfer the cuvette loaded with the samples shortly before you trigger the
pulse.
Avoid putting your finger on the aluminium electrodes or it will dramatically
increase the temperature of the sample and increase the risk of arcing.
As soon as possible (less than 30 seconds) re-suspend the cells in the
cuvette with 1ml of outgrowth medium.
Some ligation mix will need a desalting procedure prior to use (see chapter
10.4).

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

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 6


Output Voltage and Electric Field Using 1&2mm Cuvettes
Output Voltage Cuvette Used Electric Field
1 800 Volts 1mm 18 000 V.cm-1
2 500 Volts 1mm 25 000 V.cm-1
1 800 Volts 2mm 9 000 V.cm-1
2 500 Volts 2mm 12 500 V.cm-1

Optimisation Advice for Gram-Bacteria


Electrical parameters for Gram- Bacteria electroporation are well known. The
pulse time must be about 5ms 6 and the electric fields usually used are
12.5kV.cm-1 and 18.0kV x cm-1. However, if necessary, the chart below gives
the electric field range where efficient electroporation of Gram- bacteria have
been observed. For Optimisation, use 100 V increments in the window range,
12.5 kV and 18.0 kV.cm-1 as starting points.

F IGURE 01: E FFICIENT ELECTRIC FIELD “ WINDOW ” FOR THE


ELECTROPORATION OF G R A M- BACTERIA .

Optimisation Advice for Gram+Bacteria


Electrical parameters for Gram+ Bacteria electroporation extend on a wider
window. The pulse time is the same as that for gram- bacteria; about 5ms7
and the electric field may vary between 6.5 kV.cm-1 to 25kV.cm-1. However,
for most Gram+ species the standard 12.5 kV.cm-1 will give good results. For
Optimisation, use 100 V increments in the window range, 12.5 kV and 18.0
kV.cm-1 as starting points.

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

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 7


For very high electric field electroporation of Gram+ bacteria (i.e.
Staphylococcus up to 23.0 kV.cm-1) pulse timed should ideally be shorter
(2.5ms) and shunt resistor of 99 Ohms will be selected.

F IGURE 02: E FFICIENT ELECTRIC FIELDS “ WINDOW ” FOR THE


ELECTROPORATION OF G RAM BACTERIA .

General Optimisation Advice for Bacteria


The following parameters are commonly used to improve the transformation
efficiency:
Electrocompetent cell preparation including, growth of the cells, harvest
timing, harvesting temperature, vessel cleanliness and water quality.
DNA quality, desalting procedure may be required (chapter 10.4).

Timing and ice temperature.


Post electroporation events including timing and the quality of the outgrowth
medium
• Once a high efficiency batch has been prepared it should be aliquoted,
and stored at –80°C for future high efficiency demanding experiments.
Transformation efficiency of electrocompetent cells is generally stable
for a minimum of 12 months at –80°C.

Troubleshooting ~ Bacterial Electroporation


Symptom Usual Causes Corrective Action

Arcing Cuvette temperature Sample conductivity increase with


temperature. The sample and cuvette
must be kept at ice temperature. When
the cuvette is transferred into the
electroporation chamber, trigger the pulse
immediately.
Touching the aluminium electrodes with
fingers dramatically increases sample
temperature. If it is necessary to dry the
electrodes prior to electroporation do it
carefully and quickly. Condensation on

the electrodes does not interfere with the


process.

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 8


Salt concentration of the Since the cuvette is exposed to an electric
DNA solution. field of 10-18 kV.cm-1, the conductivity of
the sample must be very low to prevent
arcing. For example, a maximum of 0.3-
0.5µl of a standard ligation mixture can be
added directly to 20µl of electrocompetent
cells before arcing begins to occur.
Different methods have been described to
decrease the concentration, such as
dilution (10x), precipitation, micro-dialyse
and desalting technique (see chapter
10.4).

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 oxide will increase pulse after
pulse and will change the electrical
parameters. Steam sterilisations
dramatically speeds-up this process.
When reusing cuvettes, DNA cross
contamination should be considered as a
potentially serious problem.

Low efficiency Cell preparation When the proper OD is reached,


temperature must be decreased as
quickly as possible.
The water quality is critical. Only fresh
MilliQ® water or equivalent should be
used.
Traces of detergent dramatically decrease
the electro-competence of the
cells. Only disposable vessels should be
used wherever it is possible.
During the preparation of the cells ice
temperature must be carefully maintained.

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 9


Fast growing cells usually give the best
results.
Quality of the outgrowth medium can
significantly improve the efficiency.
DH10B should give 109 transformants per
µg of DNA routinely with a high purity
pUC19.

THERMO ELECTRON ELECTROPORATION


OPTIMISATION GUIDE

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 10


CHAPTER IV: MAMMALIAN CELL ELECTROPORATION

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.

1. Harvest the cells as normal using the standard laboratory


procedure. Do not attempt for example to reduce the trypsin
concentration or exposure time.
2. Wash the cells twice in growth medium by pelleting (50g for
10 minutes) to remove contaminating trypsin.
3. Re-suspend them in 50ml of growing medium at room
temperature.
4. Carefully count the cells. The concentration of the cells in the
electroporation medium should be carefully controlled to
obtain reproducible transfection efficiencies.
5. Pellets aliquots of 5.106cells in 1.5ml Eppendorf tubes at 50g
for 10 minutes.
6. For each sample, mix in a 1.5ml Eppendorf tube9, 30µg of
purified DNA 10 (only for the first trials, adjust the DNA
concentration), and adjust the final volume to 800µl with
growing medium or even better with OptiBuffer (Cat # EKIT-
E1).
7. Re-suspend the cells in the electroporation medium and
incubate at room temperature for 1 to 3 minutes maximum.

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.

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 11


The following advice is valid for any kind of mammalian cells:

Fast growing cells usually give better results. For cells growing adherent11,
we recommend to harvest at 50% to 70% confluence.

Operations following trypsinisation must be done in the shortest time possible.


Trypsinisation removes some of the membrane embedded proteins,
increasing membrane fluidity and easing pores resealing after the pulse, thus
usually increasing cell survival after electroporation.

Water quality is important; we recommend you only use freshly prepared


18MΩ, stored in detergent free clean vessels, or better in a disposable vessel.

All chemicals used should be dedicated to cell culture and preparation and
should be a molecular biology grade.

Standard Cell Line Electroporation


1. Transfer the cell suspension into the cuvette at room temperature.
2. Trigger the pulse immediately.

Cuvette 4mm
Voltage 250 Volts
Capacitor 1500µF
Shunt Resistor …… (Infinite)

3. Immediately (less then 30 seconds is recommended after the


pulse) transfer the cell suspension to a dish containing pre-
warmed culture medium with serum.

The following advice is valid for all mammalian cells:

DNA quality is critical. We recommend CsCl centrifugation followed by the


NaCl centrifugation. However, good quality endotoxin free purification kits can
be a suitable alternative (see chapter 10.2 and 10.3).

DNA concentration required can be relatively high. When DNA is purified as


recommended the electroporation efficiency increases linearly with the DNA
concentration.

Mammalian cell electroporation should be done at room temperature, which


increases membrane fluidity and eases pore resealing after the pulse, thus
usually increasing cell survival.

The electroporation medium can be critical for successful electroporation. We


recommend using the normal growing medium with or without serum. Thermo

11
However it is now possible to electroporate efficiently quiescent cells with the InSitu electroporation system
from Thermo

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 12


OptiBuffer, which mimics cytoplasm composition, may also greatly improve12
electroporation efficiency.

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.

Optimisation Advice for Mammalian Cell Lines


For most mammalian cell lines the optimum output voltage is 250V with 4mm
cuvette loaded with 800µl and a capacitor of 1500µF.

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.

Figure 03: Efficient output voltages “window” for the electroporation of


Mammalian cell Lines.

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.

Optimisation Advice for Primary Mammalian Cells


For most Primary mammalian cells the optimum output voltage is 300V with
4mm cuvette loaded with 800µl and a capacitor of 1500µF.

12
See Thermo Hybaid newsletter October 1997 for more details.

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 13


Below you will find the “window” of the most usual voltage output use for the
electroporation of mammalian Primary cells with 4mm cuvette and a sample
volume of 800µl.

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.

Figure 04:Efficient output voltages “window” for the electroporation of Primary


Mammalian cell.
General Optimisation Advice for Mammalian Cells
If it is necessary to improve transformation efficiencies we suggest the
procedure described below should be followed.

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.

To optimise results the following parameters are commonly used to improve


the transformation efficiency:

Electrocompetent cell preparation including growth of the cells, DNA quality,


and extreme care must be applied to the preparation of DNA. For best results
we recommend one CsCl centrifugation followed by one NaCl centrifugation.
However, good quality endotoxin free purification kits can be a suitable
alternative (see chapter 10.2 and 10.3).

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 14


Electroporation medium quality is also very important. During the process the
cells will be highly exposed to the external medium. It is recommended to use
your usual growth medium supplemented as necessary with serum or even
better, OptiBuffer.
Post electroporation events including timing and the quality of the outgrowth
medium

Troubleshooting ~ Mammalian Cell Electroporation

Symptom Usual Causes Corrective Action

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.

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 15


THERMO ELECTRON ELECTROPORATION

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 16


OPTIMISATION GUIDE

CHAPTER V: YEAST ELECTROPORATION

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.

Yeast Preparation for Electroporation


1. Inoculate 500ml YPD broth in a 2 – 1 flask. Grow with vigorous
shaking at 30°C to OD600mm = 1.3 -1.5 (approximately 1 x 1010
cells/ml).
2. Harvest the culture by centrifugation and resuspend in 100ml
YPD broth. Add 2.0ml sterile 1M HEPES, pH 8.0 (20mM final
concentration) and then add 2.5ml sterile 1M dithiothreitol (DTT;
25mM final concentration) while swirling gently. Incubate 15 min
at 30°C with gentle shaking.
3. Bring to 500ml with Milli-Q H2O.
4. Concentrate the cells approximately 1000-fold with several
centrifugations, re-suspending the successive pellets as follows:
1st pellet: 500ml Milli-Q H2 O
nd
2 pellet: 250ml Milli-Q H2 O
3rd pellet: 20ml 1M sorbitol
th
4 pellet: 0.5ml 1M sorbitol
Resuspension should be vigorous. The rotor, the speed and exact
duration of centrifuge spins are not critical. All solutions should be
ice-cold. Final volume of resuspended yeast should be about 1.0 -
1.5ml.
5. Re-suspend the cells in an appropriate volume of washing
solution so as to reach an OD600 of 400. This means that the
final volume should be 1/400 to 1/200 of the initial culture volume.
The cells can be kept on ice for several hours without any loss of
transformation frequency.

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.

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 17


3. Transfer the cuvette into the electroporation chamber and trigger
the pulse immediately.

Cuvette 2mm
Voltage 1700 Volts
Capacitor 25µF
Shunt Resistor 486R (or closest
value)

4. As soon as possible (within 30 seconds) re-suspend the cells in


the cuvette with 1ml of YPD medium (room Temperature) into the
cuvette. Incubate for 1 hour.
5. Spread 50 to 100µl aliquots onto selective plates. It takes usually
a longer time for colonies to develop after electroporation that with
traditional transformation techniques.

Optimisation Advice for Yeast


Electrical parameters for yeast electroporation extend in two different
windows. Efficient voltages are found in the low range as well as in the high
voltage range. We have chosen to describe the low voltage electroporation
methods with a 150µF capacitor. Please keep in mind that high voltage
electroporation methods also exist.
Resistor

486R

335R
1400 1500 1600 1700 1800V

Output Voltage
Figure 05: Efficient output voltage and shunt resistor “window” for the
electroporation of yeast.

General Optimisation Advice for Yeast


Voltage Optimisation is only requested if the killing rate is too high. Further
Optimisation is possible through the resistor value, which will influence the
pulse time.

To obtain maximum efficiency we recommend using the following parameters:


Electrocompetent cells preparation including, growth of the cells, harvest
timing, harvesting temperature, vessel cleanliness and water quality.

DNA quality, desalting procedure may be required and DNA purification as


describe chapter 10.4 is highly recommended for optimum results.

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 18


Timing and ice temperature during cell preparation and electroporation.
Post electroporation events including timing and the quality of the outgrowth
medium. Electroporated cells must be transferred within 30 seconds following
the pulse into fresh outgrowth medium.

Water quality is important; we recommend you only use freshly prepared


18MΩ, stored in detergent free cleanliness vessels, or better in disposable
vessel.

Chemicals grade. All chemicals used should be dedicated to cells culture and
preparation and should be of molecular biology grade.

Troubleshooting ~ Yeast Electroporation


Symptom Usual Causes Corrective Action

Arcing Cuvette temperature Sample conductivity increases with


temperature. The sample and cuvette
must be kept at ice temperature.
When the cuvette is transferred into
the electroporation chamber, trigger
the pulse immediately.
Touching the aluminium electrodes
with fingers dramatically increases
sample temperature. If it is necessary
to dry the electrodes prior to
electroporation do it carefully and
quickly. Condensation on the
electrodes does not interfere with the
process.
Salt concentration of the Since the cuvette is exposed to a
DNA solution relatively high electric field, the
conductivity of the sample must be
very low to prevent arcing. Different
methods have been described to
decrease the salt concentration, such
as dilution (10x), precipitation, micro-
dialyse and desalting technique
(chapter 10.4).

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

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 19


oxide will increase pulse after pulse
and will change the electrical
parameters. Steam sterilisations
dramatically speed-up this process.
When reusing cuvettes, DNA cross
contamination should be considered
as a potentially serious problem.
Low efficiency Cell preparation When the proper OD is reached,
temperature must be decreased as
quickly as possible.
The water quality is critical. Only
freshly prepared MilliQ water or
equivalent should be used.
Traces of detergent dramatically
decrease the electro-competence of
the cells. Only disposable vessels
should be used wherever it is
possible.
During the preparation of the cells the
ice temperature must be carefully
maintained.
Fast growing cells usually give the
best results.
Quality of the outgrowth medium can
significantly improve the efficiency.

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 20


THERMO ELECTRON ELECTROPORATION

OPTIMISATION GUIDE

CHAPTER VI: SPECIAL APPLICATIONS

Fish, birds and other eukaryotic cell electroporation


Refer to Chapter IV, Mammalian Cell Electroporation, for general advice and
starting electroporation parameters and to Chapter V, Troubleshooting for
Mammalian Cells Electroporation. Starting parameters are 250 Volts, 1500µF,
4mm cuvettes loaded with 800µl of sample. Cell concentration should be
between 5x106 to 3x107 cells.ml-1 and DNA concentration about 30µg/800µl.
The same advice applies for established cell lines and Primary cells. Primary
cells usually require higher electric field.

Cuvette 4mm
Voltage 250 Volts
Capacitor 1500µF
Shunt Resistor …… (Infinite)
Temperature Room Temp.

Standard parameters for using the optipulse option


This option should only be used after extensive testing using the single pulse.
Or if you are unhappy with the levels of transfection using the single p ulse and
wish to try and increase them. In DoublePulse mode when you trigger the
pulse by pressing the RUN key, the Cellject Pro will display a message asking
you to choose between the normal DoublePulse or OptiPulse (F2) – Choose
OptiPulse (F2) it will fully merge the 2 pulses.

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

Voltage 1 850 volts


Capacitor 1 25µF
#
Parallel resistor
486 R
1

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 21


Inter Pulse Delay 0.000
Voltage 2 100 volts
Capacitor 2 1500µF
#
Parallel resistor
486 R
2

Tissue electroporation using 10mm cuvettes


Cell Type Eukaryotic tissue slice
Growth phase at
N/A
Harvest

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

Output Voltage 450 Volts


3000µF (Pulse time 20 to 30 seconds). If the pulse time is
Capacitor shorter then 20ms, reduce the volume, until you reach the
minimum pulse time.
Infinite (Display show ……. For infinite resistor value on
Parallel resistor
CellJect Pro)

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 22


THERMO ELECTRON ELECTROPORATION

OPTIMISATION GUIDE

CHAPTER VII: INTACT PLANT ELECTROPORATION

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.

1. Legume seeds are sown individually in Deepot containers in


greenhouse potting soil mix. Seeds germinate within 7-10 days.
The plants are maintained at 25°C day/ 20°C night temperatures
and a 10-h night/ 14-h day photo -period. Electroporation is
performed on 3-4 wk-old plants depending on the legume crop.
2. Day 1. On the day preceding electroporation, the plants are
prepared by decapitation close to the node of a fully expanded
leaf. Only the most terminal nodal bud is retained and all other
axillary buds are excised (Fig. 06). The bud that is retained is
allowed to grow overnight.
3. Day 2. Plasmid DNA to be used for electroporation is mixed with a
polycationic transfection lipid13 and left at room temperature for
about 20 minutes. Sterile MS salts solution is added to the DNA-
lipofection-molecule mixture to a final volume of 2.0ml. Using a
Hamilton micro-syringe, 2µl of DNA solution are injected into the
most terminal nodal bud that was retained on the plants.
Note: The amount of lipofection molecule added to the plasmid DNA is
critical and must be optimised. We suggest, 100µL of EasyFector
with 200µg of plasmid DNA to a total volume of 800µl. EasyFector
reagent interacts spontaneously with DNA to form a lipid-DNA
complex.

13
Thermo commercialises 2 polycationic lipids: EasyFector cat. # ETR-E1 and PrimeFector cat. # ETR-P1.

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 23


F IGURE 0 6 : I NTACT PLANT BUD ELECTROPORATION .

Plant Bud Electroporation


1. The treated bud is electroporated by dipping it into 2ml
electroporation buffer (200-300µg.ml-1 of DNA+EasyFector+MS
salts solution) contained in a 10mm electroporation cuvette (Cat.
# ECU-110 and ECU-210).
2. Trigger the pulse without delay.

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.

General Optimisation Advice for Intact Plant


Although plants at any stage before flowering can be electroporated to obtain
successful transformation, electroporation of plants that are about 3-4 weeks
old increases the efficiency of stable integration.

Plasmid DNA of high purity (i.e. free of contaminating bacterial chromosomal


DNA and RNA, see chapter X) enhances the plasmid integration frequency.

Concentration of DNA in the electroporation medium is critical, 200 to


300µg.ml-1 DNA seems to be optimum.

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.

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 24


Following electroporation, although the plant is recovering and the
electroporated meristem is growing to form a branch, growth from all the
auxiliary buds below the electroporated meristem should be discouraged by
excising them regularly.

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 25


© Thermo Electron Corporation 2003 Issue 4.0, June 2003 26
THERMO ELECTRON ELECTROPORATION

OPTIMISATION GUIDE

CHAPTER VIII: PLANT PROTOPLAST ELECTROPORATION

Plant Protoplast Preparation


1. Prepare protoplast from leaves or suspension cultures by
conve ntional procedures.
2. Wash the protoplasts twice with HBS + mannitol14 and
resuspend them in HBS + mannitol at a density of 2 -4 x 106
protoplasts.ml-1.
3. Mix an equal volume of protoplasts suspension with a
solution of plasmid DNA (20µg.ml-1 dissolved in HBS +
mannitol and filter sterilised); let stand for 1 to 3 minutes
maximum.

Plant Protoplast Electroporation


1. Resuspend the protoplasts by gentle agitation, and introduce 0.8ml
of the protoplast~DNA mixture into a sterile electroporation 4mm
electroporation cuvette.
2. Trigger the pulse immediately (8-10 ms RC time constant)

Cuvette 4mm
Voltage 280 Volts
Capacitor 1050µF
Shunt Resistor 201R
Temperature Room Temp.

3. Immediately (less then 30 seconds is recommended after the


pulse.) transfer the cell in fresh outgrowth medium.
Note: dilute the protoplast 1:10 (final volume 8-ml) with
culture medium. After 15 minutes centrifuge (100g. 5
minutes) and resuspend in 2ml of culture medium. Culture
the protoplasts at usual temperature.
4. Controls should include protoplasts mixed with DNA and cultured
without being electroporated, and protoplasts electroporated in the
absence of DNA.

General Optimisation Advice For Plant Protoplast


All operations should be carried out at room temperature with sterile solutions
in a laminar flow hood.

14
0.2M to 0.6M depending on the cell type.

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 27


Water quality is important; we recommend you only use freshly prepared
18MΩ, stored in detergent free clean vessels, or better in a disposable vessel.
All chemicals used should be dedicated to cell culture and preparation and
should be a molecular biology grade.
DNA quality is critical. We only recommend the CsCl centrifugation followed
by the NaCl centrifugation (chapter 10.2 and 10.3). Other purification
techniques to prepare transfection grade DNA are slowly emerging.

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 28


THERMO ELECTRON ELECTROPORATION

OPTIMISATION GUIDE

CHAPTER IX: PROTEIN, OLIGO AND SMALL MOLECULE


ELECTROPORATION

The usual electroporation protocol should be used (see previous chapters).


However, proteins, oligonucleotides, and antibodies have a relatively small
size in comparison with DNA. In general the electric field necessary to
achieve efficient molecule transfer will be 10 to 20% lower than for the
equivalent protocol for transformation.

Cuvette 4mm
Voltage 230Volts
Capacitor 900µF
Shunt Resistor …… (Infinite)
Temperature Room Temp.

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 29


THERMO ELECTRON ELECTROPORATION

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 30


OPTIMISATION GUIDE

CHAPTER X: DNA PURIFICATION

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.

DNA concentration measured by absorbance is generally over estimated by a


factor of 2 or 3 if the DNA as been purified with a wrong technique or only the
CsCl centrifugation (double CsCl centrifugation does not improve DNA quality
for transfection). Equally, if the absorbency ratio at 260nm versus 280nm is
good (1.8), it does not prove that the DNA preparation is pure ix,x .

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,

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 31


Ti65, or Ti70 rotor (or their equivalent). Fill the remainder of the
tube with light paraffin oil and seal the tube.
6. Centrifuge the density gradients at 45,000 rpm for 16 hours
(Vti65), 45,000 rpm for 48 hours (Ti50), 60,000 rpm for 24 hours
(Ti65), or 60,000 rpm for 24 hours (Ti70.1) at 20°C.
7. Two bands of DNA, located in the centre of the gradient should be
visible in ordinary light. The upper band, which usually contains
less material, consists of linear bacterial (chromosomal) DNA and
nicked circular plasmid DNA; the lower band consists of closed
circular plasmid DNA. The deep-red pellet on the bottom of the
tube consists of ethidium bromide~RNA complexes. The material
between the CsCl solution and the paraffin oil is protein. NOTE:
CsCl-Ethidium bromide gradients in Beckman Quick-Seal tubes
can accommodate up to 4mg of closed circular plasmid DNA
without becoming overloaded. If more plasmid DNA is present, it
spreads into a wide band that overlaps with chromosomal DNA.
This problem, which arises only with plasmids that replicate to
extremely high levels, can be avoided by dividing such plasmid
preparations between two gradients. If overloading does occur,
collect the entire band of DNA, adjust the volume of the solution
to 15 ml with CsCl solution (ρ = 1.58g.ml-1), and re-centrifuge the
DNA to equilibrium in two centrifuge tubes.
8. Collect the bands of DNA. Insert a 21-gauge hypodermic needle
into the top of the tube to allow air to enter. To minimise the
possibility of contamination, first collect the upper band
(chromosomal DNA) through an 18-gauge hypodermic needle as
follows; Carefully wipe the outside of the tube with ethanol to
remove any grease or oil, and then attach a piece of Scotch Tape
to the outside of the tube. Insert an 18-gauge hypodermic needle
(bevelled side up) into the tube through the tape so that the open,
bevelled side of the needle is positioned just below the band of
chromosomal DNA and parallel to it. Collect the viscous DNA into
a disposable tube, and then seal the end of the hypodermic
needle with a plug of modelling clay. Leaving the second needle
in place, insert a third hypodermic needle (18-gauge) through the
tape and collect the lower band of plasmid DNA into a glass or
plastic tube. Remove ethidium .
Removal of RNA From Preparation of Plasmid DNA
For some it is essential to obtain DNA preparations that are free of
contaminating RNA. Although the weight of such contaminants in plasmid
DNA prepared by equilibrium centrifugation in CsCl-Ethidium bromide
gradients is small, the number of RNA molecules can be relatively large and
can contribute significantly to the total number of 5’ termini. RNA can be
removed from plasmid preparations by the following method.

This is an unpublished protocol of B. Seed.


1. Add 0.1 volume of 3M sodium acetate (pH 5.2) and precipitate the
nucleic acids with 2 volumes of ethanol for 30 minutes at 4°C.

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 32


2. Recover the nucleic acids by centrifugation at > 10,000g for 15
minutes at 4°C. Drain off as much of the supernatant as possible,
and then store the open tube on the bench to allow the last traces
of ethanol to evaporate.
3. Dissolve the pellet in TE (pH 8.0) at a concentration of at least
100µg.ml-1.
4. Add DNAse-free pancreatic RNAse (see Appendix B) to a final
concentration of 10µg.ml-1.
5. Incubate the mixture for 1 hour at room temperature.
6. Add 4ml of TE (pH 8.0) containing 1M NaCl to a Beckman
SW50.1 centrifuge tube or its equivalent). Using an automatic
pipetting device equipped with a disposable tip, layer up to 1ml of
the RNAse-treated plasmid preparation on top of the 1 M NaCl
solution. If necessary, fill the tube with TE (pH 8.0). Centrifuge at
40,000 rpm for 6 hours at 20°C in a Beckman SW50.1 rotor (or its
equivalent). The plasmid DNA sediments drop to the bottom of
the tube while the ribo-oligonucleotides remain in the supernatant.
7. Discard the supernatant. Re -dissolve the pellet of plasmid DNA in
5.0ml of TE (pH 8.0). Add 50µl of 3M sodium acetate and recover
the DNA by precipitation with 2 volumes of ethanol for 10 minutes
at 4°C and centrifugation at >10,000g for 15 minutes at 4°C.
Desalting of Ligation Mix
This method has been published in BioTechniques 21:1024 (December 1996)
by Alexey M. Atrazhev and John F. Elliott.

Since the cuvette is exposed to an electric field of 10-18 kV/cm, the


conductivity of the sample must be very low to prevent arcing. For example, a
maximum of 0.3-0.5µl of a standard ligation mixture (typically 10-50µl total
volume) can be added directly to 20µL of electrocompetent cells before arcing
begins to occur. Various protocols have beep proposed to desalt DNA in a
small volume, including: traditional ethanol-precipitation, rinsing and re-
suspension; gel filtration on micro-columns or drop dialysis through
membranes.

This method requires minimal manipulation of the DNA, which is a significant


advantage for ligation involving large DNA fragments.

1. Fill an Eppendorf tube with 1.5ml of molten 1% agarose in 100mM


glucose.
2. Before gelling, vertically immerse a 200µl plastic micropipette tip
into the surface of the agarose through a pre-formed hole in the
lid (created for example by drilling a small hole).
3. After the agarose solidifies remove the tip to leave a conical
shaped well of approximately 30-l00µl volume, depending on how
far the tip has been immersed into the agarose.
4. Load the completed ligation reaction into the "well" using a
micropipette and incubated for 90 minutes on ice to let the salt
diffuse into the agarose.

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 33


5. Carefully remove the liquid mixture from the well, and a portion of
it is directly electroporated into bacteria.

THERMO ELECTRON ELECTROPORATION


OPTIMISATION GUIDE

CHAPTER XI: DOUBLE & OPTIPULSE

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.

Figure 2: Single pulse and DoublePulse electroporation.

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 34


Energy Level Single Pulse versus DoublePulse
Energy levels used can be calculated using the following formula:
P=0.5 x CV2
Single Pulse:
Typical parameters = 250 volts and 1000µF
P= 0.5 x 1000.10-6 x 2502 = 31.25 Joule
DoublePulse:
Typical parameters = 750 volts and 25µF & 100 volts and 1500µF
P= (0.5 x 25.10-6 x 7502) + (0.5 x 1,5.10-3 x 1002) = 14.53 J
From these figures it can clearly be seen that the energy level used from this
example is 50% of a single pulse method.

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.

Figure 5: OptiPulse discharge electroporation

T HE CURRENT THEORY IS THAT THIS FAST DISCHARGE PROCESS REDUCES


ANY PORE RE - SEALING THAT MAY OCCUR BETWEEN THE DOUBLE PULSES ,
POSSIBLY IMPROVING T HE OVERALL EFFICIENCY .

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 35


THERMO ELECTRON ELECTROPORATION
OPTIMISATION GUIDE
CHAPTER XII: MEDIUM AND BUFFERS

LB Medium (Luria -Bertani Medium)


Per Litre: To 950ml of de-ionised H2 O, add:
Bacto-tryptone 10g
Bacto-yeast extract 5g
NaCl 10g

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

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 36


sugar has dissolved, adjust the volume of the solution to 100ml with de-
ionised H2O and sterilise by filtration through a 0.22-micron filter).
2x HEPES-Buffered Saline
Dissolve 1.6g of NaCl, 0.74g of KCl, 0.027g of Na 2HPO4·2H2O, 0.2g of
dextrose, and 1g of HEPES in a total volume of 90ml of distilled H2 O. Adjust
the pH to 7.05 with 0.5N NaOH, and then adjust the volume to 100ml with
distilled H2 O. Sterilise the solution by passage through a 0.22-micron filter.
Store in 5ml aliquots at -20°C.
Phosphate-Buffered Saline (PBS)
Dissolve 8g of NaCl, 0.2g of KCl, 1.44g of Na 2 HPO4, and 0.24g of KH2PO4 in
800ml of distilled H2 O. Adjust the pH to 7.4 with HCl. Add H2O to 1 litre.
Dispense the solution into aliquots and sterilise them by autoclaving for 20
minutes at 15lb/sq. in. on liquid cycle. Store at room temperature.
1M Tris
Dissolve 121.1g of Tris base i n 800ml of H2O. Adjust the pH to the desired
value by adding concentrated HCl.

pH HCl
7.4 70ml
7.6 60ml
8.0 42ml

Allow the solution to cool to room temperature before making final


adjustments to the pH. Adjust the volume of the solution to 1 litre with H2 O.
Dispense into aliquots and sterilise by autoclaving. Note: If the 1M solution
has a yellow colour, discard it and obtain better quality Tris. Although many
types of electrodes do not accurately measure the pH of Tris solutions,
suitable electrodes can be obtained from most manufacturers. The pH of Tris
solutions is temperature-dependent and decreases approximately 0.03 pH
units for each 1°C increase in temperature. For example, a 0.05M solution
has pH values of 9.5, 8.9 and 8.6 at 5°C, 25°C, and 37°C, respectively.
TE
pH 7.4
10mM Tris · Cl (pH 7.4)
1mM EDTA (pH 8.0)

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)

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 37


STET
0.1M NaCl
10mM Tris · Cl (pH 8.0)
1mM EDTA 9pH 8.0)
5% Triton X-100
TNT
10mM Tris · Cl (pH 8.0)
150mM NaCl
0.05% Tween 20

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 38


THERMO ELECTRON ELECTROPORATION

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 39


OPTIMISATION GUIDE

CHAPTER XIII: GENERAL INFORMATION

The Cleanliness of the Glassware and Plasticware.


Because the presence of trace amounts of detergent or other chemicals
greatly reduces the efficiency of transformation, it is best to set aside a batch
of glassware that is used for no other purpose than transfection. This
glassware should be washed and rinsed by hand, filled with pure water (MilliQ
or equivalent), and sterilised by autoclaving. The water should be discarded
just before the glassware is used.
Transfection Principle
This is the delivery of foreign molecules such as DNA or RNA into living cells.
This technique has become a powerful tool for the study and control of gene
expression. Different transfection techniques can be used; transient
transfection and stable transfection.
Transient Transfection
When cells are transiently transfected, the DNA is introduced into the cell, but
does not integrate into the chromosome. This means that many copies of the
gene can be present. Transcription of the reporter gene can be analysed
within 24 to 96 hours after introduction of the DNA. Transient transfection is
most efficient when supercoiled plasmid DNA is used.
Stable Transfection
With stable transfection, the transfected DNA is either integrated into the
genome or maintained as an episome. Stable transfection involving
integration of the DNA is most efficient when linearised plasmid DNA is used,
since linearisation facilitates recombination of the DNA with the cell
chromosome. Cells that have successfully integrated the DNA of interest can
be distinguished by using detectable markers. Frequently used detectable
markers are the genes encoding aminoglycoside phosphotransferase (APH;
neor gene) or hygrornycin B phosphotransferase (HPH). Other detectable
markers are the genes encoding adenosine deaminase (ADA), dihydrofolate
reductase (DHFR), thymidine kinase or xanthine-guanine phosphoribosyl
tranferase (XGPRT; gpt gene).
Primary Cells and Cell Lines
Cells or cell lines vary greatly with respect to their growth behaviour and
nutritional requirements. Optimisation of the cell culture work is therefore
necessary to ensure that cells are healthy and in Prol condition for
transfection.

Primary Cell Culture


Primary cell cultures arise from the outgrowth of migrating cells from a piece
of tissue or by enzymatic, chemical, or mechanical dispersal of the tissue.
Primary cell cultures are morphologically most similar to the parent tissue.

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 40


Finite Cell Line
Finite cell lines are formed after the first sub-culturing of a Primary cell culture,
and can be propagated and sub-cultured several times.
Established Cell Line
There is a limit to the number of generations that a finite cell line can be
propagated. After that it will either die out or acquire a stable, heritable
alteration, giving rise to an established cell line (i.e.: Hela cells).
Adherent and Suspension Cells
Cell cultures or cell lines grow as an adherent layer(s) or in suspension
depending on their origin.
Adherent Cells
Adherent cells are anchorage-dependent and propagate as a mono or multi
layer(s) attached to the culture vessel. This attachment is essential for
proliferation. Most cells derived from tissues are anchorage-dependent.
Suspension Cells
Suspension cells are able to survive and proliferate without attachment.
Hematopoeïtic cells, transformed cell lines, and cells from malignant tumours
can be grown in suspension.
Media and Supplements
Media are composed of a mixture of essential salts, nutrients, and buffering
agents. Alternatively, packaged premixed powders are available. Most media
purchased are guaranteed to be mycoplasma and endotoxin free.
Supplements to the media must include glutamine and can include non-
essential amino acids, sodium pyruvate and antibiotics. Some common media
include F12, DMEM, RPMI 1640, DMEM/F12, MEM, S-MEM.
Serum
In most cases media are supplemented shortly before use with serum. Foetal
calf serum (FCS) is often used, but for some applications less expensive sera
like horse- or calf serum can be used. Generally serum is a partially undefined
material, which contains growth and attachment factors and may show
considerable variation in the ability to support growth of particular cells.
Variations in the serum quality can also lead to variation in transfection
efficiency. In general, it is advisable to test a small batch of serum from a
reputable supplier with a control cell line and assay before performing
transfection experiments. Once a given batch has been shown to yield
satisfactory and reproducible results, additional sera from the same lot should
be purchased.
Transfection Methods
Of the variety of different transfection methods, DEAE-dextran, calcium
phosphate, electroporation, and liposome-mediated transfection are the most
commonly used. Each individual method has its characteristic advantages and
disadvantages and the choice of transfection method strongly influences
transfection results. If you are not sure of the most adequate technique do not
hesitate to contact us for some advice.

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 41


Plasmid DNA Quality
The quality of the plasmid DNA STRONGLY influences the results of
transfection experiments. Therefore only plasmid DNA of the highest quality,
which is completely free of contaminating RNA, genomic DNA or proteins,
should be used. We consider that DNA purified as described in chapter 10
gives the best results.
Conformation and Concentration of DNA
Although both linear and circular DNA can be transfected by electroporation,
higher levels of stable transformation are obtained when linear DNA is used.
Effective transfection has been obtained with concentrations of DNA ranging
from 1µg/ml to 80µg/ml.
Genetic Reporter Systems
After cloning a gene of interest, transfection is a useful tool to determine how
cis-acting sequences, such as promoters and enhancers, and trans-acting
factors, such as transcription factors, act together to control eukaryotic gene
expression. The reporter gene provides an indirect way of measuring how
such regulatory sequences influence gene expression. Reporter genes are
also useful in serving as controls. Transfection efficiencies between
transfection experiments can be standardised by comparing the expression of
the reporter gene product.

In choosing a suitable reporter system, several considerations should be


taken into account. First, the reporter gene should be absent from the cells
used in the study or easily distinguished from the native form of the gene.
Second, the assay for the reporter gene product should be quick, easy,
sensitive, and inexpensive. In particular, a broad linear range is important to
enable detection of both small and large changes in the reporter gene
expression. Finally, the presence of the reporter gene should not affect the
physiology of the cells being used.

Commonly Used Reporter Genes


Chloramphenicol Acetyltransferase
The prokaryotic enzyme chloramphenicol acetyltransferase (CAT) is
commonly used as a reporter. This enzyme catalyses the transfer of acetyl-
groups from acetyl-coenzyrne-A to chloramphenicol. In the common CAT
assay, cell lysates prepared from transfected cells are incubated with 14C-
Iabeled chloramphenicol. The resulting acetylated and unacetylated forms of
chloramphenicol are separated by thin-layer chromatography. A qualitative
estimate of CAT activity can be obtained simply by exposing the plates to X-
ray film. For quantitative analysis, the separated bands can be scraped from
the thin-layer plate and the levels of radioactivity measured in a scintillation
counter.
Firefly Luciferase
Luciferase catalyses a bioluminescent reaction involving the substrate
luciferin, ATP, Mg2+ , and molecular oxygen. When these components are

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 42


mixed with cell lysates containing luciferase, a flash of light is emitted. Light
signals are detected using a luminometer or a liquid scintillation counter.
B-Galactosidase
The prokaryotic enzyme B-galactosidase can be assayed calorimetrically
using the substrate o-nitrophenyl-3-D-galactopyranoside (ONPG). The
hydrolysis of ONPG by B-galactosidase yields a yellow-coloured product, o -
nitrophenol, which can be measured photometrically.
Human Growth Hormone (HGH)
The assay for human growth hormone is based on immprimalogical detection
of hGH secreted by transfected cells. Specific 125 I-Iabeled antibodies against
hGH are used and results are monitored in a scintillation counter.
Green Fluorescent Protein
Green fluorescent protein (GFP), originally isolated from the jellyfish Aequorea
Victoria, has the ability to absorb blue light and emit green light. This unique
protein can be expressed in mammalian cells and protein expression can be
visually monitored in living cells. Although the system provides a convenient
way to detect protein e xpression without a specific assay, quantitative
analysis is limited. This reporter gene system is best suited for InSitu
detection of gene expression, such as localisation studies of fusion proteins
within cells.

Other Reporter Systems


• Secreted alkaline phosphatase
• 8-glucuronidase (GUS)

THERMO ELECTRON ELECTROPORATION


OPTIMISATION GUIDE

CHAPTER XIV: ELECTRICAL BASICS

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).

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 43


V is the output voltage (Volt) of the electroporation apparatus and d is the
distance (centimetre) between the electrodes of the electroporation cuvette.
Capacitor
Electronic components that usually look like a canister inside of which it is
possible to store electrical charges (electrons). The unit to measure the
capacity of a capacitor is called Farad (F). Practically all electroporation
capacitor values will vary from a few µF to a couple of thousands µF.
Pulse Energy
The energy (P) stored in a capacitor can be calculated with the following
formula:

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)].

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 44


F I G U R E 07: E XPON E N T I A L DECAY PULSE; PU L S E TIME

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.

Basic Designs for Low Voltage Application

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.

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 45


Total Resistance and Pulse Time
As stated at § 3.3 for Bacteria electroporation the voltages used are extremely
high, 2.5kV and up, resulting in very high pulse energy. These energy levels
are incompatible with cell survival, this is the reason why electroporation
systems for high voltage application includes a shunt resistor, also named
pulse time controller. Because the value of the shunt resistor are very low in
comparison with the sample resistance, most of the energy will flow through
them protecting the sample and as a consequence regulating the pulse time.

The total resistance of a circuit including shunt resistor can be calculated


using the following formula:
R-1 = R -1s + R -1L

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.

Basic Design for High Voltage Application

F IGURE 0 9 : ELECTROPORATION S YSTEM; BASIC DESIGN FOR


H IGH VOLTAGE ELECTROPORATION. A SHUNT RESISTOR IS
INSERTED.

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 46


This is the design used for high voltage electroporation, typically for Bacteria
cells, and in general for prokaryotic cell. In this type of experiment the
capacitor (C) is discharged through the sample (RL) and a shunt resistor (Rs ).

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).

THERMO ELECTRON ELECTROPORATION


OPTIMISATION GUIDE
CHAPTER XV: ORDERING INFORMATION

Electroporation Equipment
EP EJ 002 Cellject PRO

EP EJ 003 Cellject duo

EP EJ 004 Cellject Uno

EP ES 900 External remote keypad


Electroporation Accessories
EP ES 002 Standard Cuvette Chamber for 1, 2, 4 & 10 mm Cuvettes
for the CellJect Pro, CellJect Prima, CellJect Plus & One

EP ES 001 Smart Card

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 47


EP ES 100 Printer for CellJect Family

EP ES 004 Printer Ribbon for CellJect Printer (1)

EP ES 007 Paper for Printers - CellJect

Electroporation Cuvettes & Buffer

EP ECU 101 50 Sterile Individually wrapped 1mm cuvettes

EP ECU 102 50 Sterile Individually wrapped 2mm cuvettes

EP ECU 104 50 Sterile Individually wrapped 4mm cuvettes

EP ECU 110 50 Sterile Individually wrapped 10mm cuvettes

EP ECU 201 25 Sterile individually wrapped 1mm cuvettes +


25 Sterile individually wrapped disposable plastic pipettes
EP ECU 202 25 Sterile individually wrapped 2mm cuvettes
25 Sterile individually wrapped disposable plastic pipettes
EP ECU 204 25 Sterile individually wrapped 4mm cuvettes
25 Sterile individually wrapped disposable plastic pipettes
EP ECU 210 25 Sterile individually wrapped 10mm cuvettes
25 Sterile individually wrapped disposable plastic pipettes
EP ECU 300 Sterile Individually Wrapped Disposable Plastic Pipettes
for 2, 4 & 10mm Cuvettes(50/box)
EP EKIT E1 OptiBuffer Kit for Eukaryotic Cells (Min. 24 Experiments)

Lipofection Products

EP ETR E1 1ml of 1mg/ml EasyTrans reagent supplied in sterile


suspension of H2Owith complete Technical Information

EP ETR N1 1ml of 1mg/ml NovaTrans reagent supplied in sterile


suspension of H2O with complete Technical Information

EP ETR P1 1ml of 1mg/ml PrimeTrans reagent supplied in sterile


suspension of H2O with complete Technical Information

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.

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 48


v
Sukharev S.I., Klenchin V.A., Serov S.M., Chernomordik L.V., Chizmadzhev Yu A. (1992), Electroporation and
electrophoretic DNA transfer into cells. The effect of DNA interaction with electropores. Biophys J 63 (5), 1320-1327.

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.

© Thermo Electron Corporation 2003 Issue 4.0, June 2003 49