Mirus Ingenio EZporator MTR 51000 Operational manual

Ingenio® EZporator®
Electroporaon System
Product User Manual
MIR 51000
RevB 121520
mirusbio.com

Page 2
Ingenio® EZporator® Electroporaon System
Product User Manual | Instrucons for MIR 51000
Table of Contents
Technology Overview..................................................................... 3
System Components and Features.................................................. 4
Technical Specicaons.................................................................. 5
Safety Informaon—Please Read Carefully..................................... 6-7
Set-up Instrucons
Installaon.................................................................................. 8
Inializing.................................................................................... 8
Instrument Features and Controls................................................ 8
Display and Status Opons.......................................................... 9
Post-pulse Display....................................................................... 10
Voltage Adjust Knob, Pulse Buon………....................................... 10
EZporator® System Protocols
Quick Reference Guidelines......................................................... 11
Instrucons for Use with Ingenio® Soluon and Kits..................... 12-13
Opmizaon
Cell Density and Passage Number................................................ 14
Nucleic Acid Purity and Concentraon......................................... 14
Pulse Condions.......................................................................... 15
Pulse Condions for Common Cell Types …………….......................... 16
Instrument Troubleshoong........................................................... 17
Experimental Troubleshoong
Low Electroporaon Eciency..................................................... 18
High Cellular Toxicity................................................................... 19
Maintenance.................................................................................. 20
Warranty
General Warranty........................................................................ 21
Out of Warranty Service.............................................................. 22
Mirus Bio LLC
www.mirusbio.com │ [email protected] │ U.S. Toll Free: 844.MIRUSBIO (844.647.8724)│ Direct: +1.608.441.2852

Page 3
Mirus Bio LLC
www.mirusbio.com │ [email protected] │ U.S. Toll Free: 844.MIRUSBIO (844.647.8724)│ Direct: +1.608.441.2852
Ingenio® EZporator® Electroporaon System
Product User Manual | Instrucons for MIR 51000
Technology Overview
Electroporaon is a physical transfecon method that ulizes short electrical pulses
to create transient membrane pores in cells through which small parcles (e.g. DNA,
RNA, proteins, etc.) can pass. Opmal electroporator pulse sengs and composion
of cell resuspension soluon greatly enhance electro-permeabilizaon and promote
passage of parcles through the cell membrane. Since electroporaon is an eecve
transfecon method regardless of the type of molecule delivered or the target cells,
this technique provides a robust and universal approach for mulple applicaons,
including gene expression, RNAi, stable cell line generaon and CRISPR/Cas genome
eding.
Electroporaon is an invaluable alternave for cell types that are not responsive to
chemical transfecon and oen yields signicantly higher transfecon eciency.
Opmizing parameters such as nucleic acid amount, cell density and voltage for
each cell type will improve transfecon eciency and decrease cell mortality, thus
making electroporaon suitable for transfecng various cell types with any type of
nucleic acid or small molecule.
The Ingenio® EZporator® Electroporaon System was developed with both eciency
and simplicity for the researcher in mind. This system ulizes an exponenal decay
pulse* to eecvely deliver cargo to cells in a cuvee with the push of a buon. The
Ingenio® EZporator® was designed for use with Ingenio® Electroporaon Kits and
Soluon. Use of electroporaon soluons not manufactured by Mirus Bio may result
in low transfecon eciency and high cellular cytotoxicity. Addionally, use of in-
compable electroporaon soluons may cause damage to the instrument and will
void the warranty. More informaon on Ingenio® Electroporaon Soluon and Kits
is available at mirusbio.com/ingenio.
*In exponenal-decay pulse condions, a set voltage is released from the capacitor and decays
exponenally. See Opmizaon Secon (page 14) for addional details.
Ingenio® EZporator® Electroporaon System and Ingenio®
Electroporaon Soluon, cuvees and cell droppers.

Page 4
System Components and Features
The EZporator® Electroporaon System was designed for mammalian and insect cell
transfecon with Ingenio® Electroporaon Kits and Soluon.
System features include:
• Digital Liquid Crystal Display (LCD) user interface
• Pulse Generator to deliver exponenal decay pulse at user-determined voltage.
NOTE: In exponenal decay pulse condions, a set voltage is released from the
capacitor and decays exponenally.
• Cuvee Chamber compable with 0.1 cm, 0.2 cm or 0.4 cm cuvees
• Power cords with Type B, F and G electrical outlet plugs
• Low Voltage (LV) mode
- Voltage range of 20 to 400 V with 2 V resoluon
- 150 Ω internal resistance and 1,050 µF capacitance
• High Voltage (HV) mode
- Voltage range of 30 to 2,500 V with 10 V resoluon
- 150 Ω internal resistance and 36 µF capacitance
• Streamlined experimental approach: (1) Inialize instrument, (2) Set voltage
and mode with the Voltage Adjust knob and (3) Deliver electroporaon pulse
by pressing the Pulse buon.
• Dual monitoring and display of peak output voltage and me constant
System Components:
Upon receipt, carefully open the box containing the EZporator® Electroporaon
System and verify that the box contains the following:
• EZporator® Pulse Generator (1)
• EZporator® Cuvee Chamber (1)
• Power cords (3)
Mirus Bio LLC
www.mirusbio.com │ [email protected] │ U.S. Toll Free: 844.MIRUSBIO (844.647.8724)│ Direct: +1.608.441.2852
Ingenio® EZporator® Electroporaon System
Product User Manual | Instrucons for MIR 51000
EZporator® Pulse Generator Power Cords
EZporator® Cuvee Chamber

Page 5
Ingenio® EZporator® Electroporaon System
Product User Manual | Instrucons for MIR 51000
Display Type: 16-character LCD backlit
Dimensions (W×D×H) 254 mm × 254 mm × 140 mm (Pulse Generator)
Power Source
Voltage 100 to 240 VAC, 50-60 Hz, CAT I
Power 800 W
Fusing 2.5 A, T rang 250 V
Environmental Usage:
Operang Temperature 10°C to 40°C (NOTE: Intended for indoor use only)
Cooling Convecon through metal case
Relave Humidity Avoid excessively humid working environments
Altude < 2,000 m above sea level (operang)
Mechanical Characteriscs:
Maximum Voltage Output: 2,500 V peak
Maximum Pulse Length 125 ms at 400 V peak or 5 ms at 2,500 V peak
Pulse Waveform Exponenal decay
Resistance 150 Ω internal resistor
Polluon Degree 2 Not to be operated in conducve pollutants atmosphere
Ordering Informaon:
Catalog # Product
MIR 51000 Ingenio® EZporator® Electroporaon System
MIR 51100 Ingenio® EZporator® Pulse Generator
MIR 51200 Ingenio® EZporator® Cuvee Chamber
Mirus Bio LLC
www.mirusbio.com │ [email protected] │ U.S. Toll Free: 844.MIRUSBIO (844.647.8724)│ Direct: +1.608.441.2852
Technical Specicaons

Page 6
Mirus Bio LLC
www.mirusbio.com │ [email protected] │ U.S. Toll Free: 844.MIRUSBIO (844.647.8724)│ Direct: +1.608.441.2852
Ingenio® EZporator® Electroporaon System
Product User Manual | Instrucons for MIR 51000
General Safety Summary
The EZporator® Electroporaon System is safe to use when operated in accordance
with this manual. Please read the following safety precauons to ensure proper use,
as this system is designed to deliver high voltage electrical pulses. If the equipment
is improperly used, the protecon provided by the equipment may be impaired.
To prevent hazard or injury, take the following precauons:
• USE PROPER POWER CORD: Use only the power cord specied for this product
and cered for the country of use.
• USE PROPER FUSE: Use only 5 mm × 20 mm, 2.5 A, 250 V type T (me delay) fuses
with this product.
• ORIENT THE EQUIPMENT PROPERLY: Do not orient the equipment such that it
is dicult to manage the connecon and disconnecon of devices.
• ENSURE PROPER VENTILATION: Allow 3 to 5 cm of clearance on all sides of the
module for proper cooling.
• GROUND THE PRODUCT: This product is grounded through the grounding con-
ductor of the power cord. To avoid electric shock, the grounding conductor
must be connected to earth ground. Before making any connecons to the
input or output terminals, ensure that the EZporator® is properly grounded.
• OBSERVE ALL TERMINAL RATINGS: Review the user manual to learn the rangs
on all connecons.
• AVOID CONTACT WITH INTERNAL CIRCUITRY: Do not open the product or
touch any electronic circuitry inside of the product.
• AVOID EXPOSURE TO EXCESS MOISTURE: Do not expose the device to a hu-
midied environment. If uid is spilled on or near the EZporator® instrument,
performance and safety may be compromised.
• DO NOT OPERATE IF DAMAGED: If damage is suspected on or to the EZpora-
tor® Pulse Generator or Cuvee Chamber, contact qualied service personnel
to perform an inspecon prior to use.
• OBSERVE ALL WARNING LABELS ON PRODUCT: Read all labels on product to
ensure proper usage.
• ENVIRONMENTAL USAGE: This product should not be used in the presence of a
ammable atmosphere such as an anesthec mixture with air, oxygen or ni-
trous oxide.

Page 7
Ingenio® EZporator® Electroporaon System
Product User Manual | Instrucons for MIR 51000
General Safety Summary (Connued)
High Voltage Risk
This symbol indicates a risk of electric shock. Electric shocks are dangerous
and may cause personal injury or death.
This instrument contains a high voltage power supply adjustable to 2,500 V. This
presents a serious risk of personal injury if not used in accordance with design and/
or use specicaons, if used in applicaons or with products for which they are not
intended or designed, or if they are used by untrained or unqualied personnel.
Please take special note of the following:
• The user must read this manual carefully before operang the instrument.
• Removing the EZporator® Pulse Generator cover and breaking the “Warranty
Void if Seal Broken” scker will void the warranty.
• Power o the instrument before connecng or disconnecng any cords/cables.
• Do not open the Cuvee Chamber or aempt to touch the electrodes while the
electrical pulse is being applied.
• All service must be performed by Mirus Bio authorized personnel only.
If there are any quesons about the operaon of this instrument, call Mirus Bio
Technical Support at 888.530.0801 or +1.608.441.2852.
Cauon Noce
This symbol indicates that cauon is advised. Proper set-up, usage and PPE
when using this instrument are required.
The Ingenio® EZporator® Electroporaon System is intended for laboratory use only
and can be used in research and development applicaons. These systems have
been designed to meet the standards for electromagnec compability (EMC) in-
tended for laboratory equipment applicaons as well as the applicable safety re-
quirements for electrical equipment for measurement, control and laboratory use.
The unit itself does not generate waste, but may be used to treat samples that are
hazardous. Please use appropriate PPE and ensure disposal in accordance with local
regulaons and pracces.
This product should NOT be used in the presence of a ammable atmosphere such
as an anesthec mixture with air, oxygen or nitrous oxide.
This product is for RESEARCH USE ONLY and is not intended for clinical use on
animals or human paents.
Mirus Bio LLC
www.mirusbio.com │ [email protected] │ U.S. Toll Free: 844.MIRUSBIO (844.647.8724)│ Direct: +1.608.441.2852

Page 8
Mirus Bio LLC
www.mirusbio.com │ [email protected] │ U.S. Toll Free: 844.MIRUSBIO (844.647.8724)│ Direct: +1.608.441.2852
Ingenio® EZporator® Electroporaon System
Product User Manual | Instrucons for MIR 51000
Set-up Instrucons
Installaon
Place the EZporator® Pulse Generator on a laboratory bench or work table. Be sure
to allow 3 to 5 cm of clearance on all sides of the module for proper cooling. When
selecng a workspace, choose a stable, dry locaon with an easily accessible power
outlet. NOTE: It is normal for the EZporator® Pulse Generator module to be slightly
warmer than its operang environment.
Connect the power cord to the EZporator® Pulse Generator module AC Input on the
back panel, then insert the power cord plug into an appropriate electrical outlet (i.e.
110 - 240 VAC).
To connect the EZporator® Cuvee Chamber to the Pulse Generator, insert the color
coded plugs aached to the Cuvee Chamber into the HV Output located on front
panel of the EZporator® Pulse Generator.
Inializing
1. Turn the EZporator® Pulse Generator on by pushing the green power buon
located at the back of the instrument (see page 19). NOTE: Push the power
buon once to turn on. Push again to turn o. Do not hold the power buon.
2. The EZporator® will go through a series of self-test algorithms to test generator
funconality. The display will ash “EZporator®” and the soware version (e.g.
V1.01) prior to aaining a ready status.
3. When the instrument is inialized for the rst me, the display will read
“READY LV 400V.”
Instrument Features and Controls:
Cuvee Chamber High Voltage
Output
LCD
Pulse Buon
Voltage Adjust

Page 9
Ingenio® EZporator® Electroporaon System
Product User Manual | Instrucons for MIR 51000
Set-up Instrucons (Connued)
Display
Once inialized, the EZporator® Pulse Generator display will show the operaonal
status, voltage mode (i.e. LV or HV) and set voltage. Once a pulse is delivered to the
cuvee containing cells, the display will read the peak output voltage delivered and
the me constant.
Status opons include:
Ready Mode
The EZporator® is operaonal and ready to deliver a pulse. The
LCD display will show HV for the high voltage mode and LV for
low voltage mode. NOTE: Ensure that the correct voltage mode
is selected for your cell type and electroporaon buer. Use LV
mode for mammalian cell types in Ingenio® Soluon.
• LV mode: from 20 to 400 V (2 V increments), 1050 µF capacitance
• HV mode: from 20 to 2500 V (10 V increments), 6 µF capacitance
Inializing and Charging
Inializing and charging capacitor to the preset voltage level.
Pulsing
Delivering a voltage pulse.
Feedback
Displays peak voltage and me constant for delivered pulse.
Sequence Aborted
Aborted delivery of a pulse at the user’s request. To abort a
pulse sequence, press the Pulse Buon a second me before
delivery of the pulse.
SCR Failure
The instrument has not detected an output pulse during the
pulsing operaon. As a safety precauon, this message can only
be reset by turning the power o.
Charge Failure
The capacitors failed to charge properly. To reset this message,
press the ‘Pulse’ buon.
Setpoint Too Low
The entered voltage setpoint is below the normal operang
specicaons. No pulse has been applied.
Mirus Bio LLC
www.mirusbio.com │ [email protected] │ U.S. Toll Free: 844.MIRUSBIO (844.647.8724)│ Direct: +1.608.441.2852

Page 10
Ingenio® EZporator® Electroporaon System
Product User Manual | Instrucons for MIR 51000
Set-up Instrucons (Connued)
Post-pulse Display
The EZporator® instrument will display the peak discharge voltage (VP) in volts (V)
and the me constant (t) in milliseconds (ms). It is normal for the measured voltage
to dier slightly from the setpoint. This is due to internal resistance and will uctu-
ate with electrical load variaons (i.e. buer conducvity). Following the delivery of
a pulse, press the Pulse Buon or rotate the Voltage Adjust knob to return to the
ready mode.
NOTE: With exponenal decay generators such as the EZporator®, it is necessary to
idenfy and monitor the me constant (t), as this variable is dependent on the con-
ducvity and osmolarity of the electroporated sample. Signicant changes in the
electroporaon sample (e.g. variaons in cell density, volume of nucleic acid added,
electroporaon soluon used) will alter the me constant.
Voltage Adjust Knob
The EZporator® Voltage Adjust knob controls set voltage AND voltage mode (i.e. LV
and HV modes). There is no separate voltage mode switch. Use the Voltage Adjust
knob to adjust the voltage from 20 to 400 V in LV mode (in 2 V increments) and from
30 to 2,500 V in HV mode (in 10 V increments). If in LV mode, increasing of the volt-
age setpoint past 400 V will result in a switch to HV mode. The voltage then read-
justs to 0 V and connues up to 2,500 V. If in HV mode, decreasing the voltage set-
point past 0 V will result in a switch to LV mode. The voltage then readjusts to 400 V.
NOTE 1: The EZporator® emits a sound when switching between LV and HV modes.
NOTE 2: The Voltage Adjust knob is speed sensive. A quick rotaon increases the
rate of change of the voltage setpoint.
NOTE 3: LV mode is used for mammalian and insect cell types.
Pulse Buon
The Pulse buon is acvated in ‘READY’ mode. Once pressed, the generator will
bleed o the capacitors to the preset voltage, prior to delivering the pulse. The
capacitor bleed me is typically between 1-6 seconds. A pulse sequence may be
aborted by pressing the Pulse buon a second me before delivery of the pulse.
Following the delivery of a pulse, press the Pulse buon or rotate the Voltage Adjust
knob to return to the ‘READY’ mode.
Mirus Bio LLC
www.mirusbio.com │ [email protected] │ U.S. Toll Free: 844.MIRUSBIO (844.647.8724)│ Direct: +1.608.441.2852

Page 11
Ingenio® EZporator® Electroporaon System
Product User Manual | Instrucons for MIR 51000
EZporator® System Protocols
Quick Reference Guidelines
1. Connect the EZporator® Cuvee Chamber to the Pulse Generator instrument
by inserng the black and red color-coded leads of the Cuvee Chamber into
the Output ports on the front panel of the Pulse Generator.
2. Press the power switch on the back panel to inialize the EZporator®.
3. Prepare samples by combining target cells and molecules in an electroporaon
buer. Pipee an appropriate volume of the prepared sample into a 0.1 cm,
0.2 cm or 0.4 cm cuvee.
NOTE: We recommend using the Ingenio® Electroporaon Soluon and Kits for
mammalian and insect cell electroporaons.
4. Verify that the EZporator® is in ‘READY’ mode. Then use the Voltage Adjust
knob to dial in the appropriate voltage and mode (LV or HV) for the cell type
and buer to be electroporated. NOTE: LV mode is used for mammalian and
insect cell types.
5. Place the cuvee in the Cuvee Chamber and close the safety dome.
6. Press the Pulse buon. The EZporator® will charge and then deliver the
electroporaon pulse. A sound will indicate that the pulse was delivered.
NOTE: To abort a pulse, press the Pulse buon during the ‘CHARGING’ interval
before the pulse is delivered.
7. Record the peak voltage (Vp) and me constant (t).
8. Process the sample as required for the experiment.
9. Following the delivery of a pulse, press the Pulse buon or rotate the Voltage
Adjust knob to return to the ‘READY’ mode.
10. Repeat steps 5-9 for addional samples.
Mirus Bio LLC
www.mirusbio.com │ [email protected] │ U.S. Toll Free: 844.MIRUSBIO (844.647.8724)│ Direct: +1.608.441.2852

Page 12
Ingenio® EZporator® Electroporaon System
Product User Manual | Instrucons for MIR 51000
EZporator® System Protocols (Connued)
Instructions for Use with Ingenio® Electroporation Solution and Kits
The Ingenio® Electroporation Solution and Kits provide a universal, high efficiency,
low toxicity solution for electroporating nucleic acids (i.e. DNA, RNA, oligonucleo-
tides, etc.) or other molecules into mammalian and insect cell types.
Transient plasmid DNA or siRNA electroporaon protocol
A. Plate cells
1. Approximately 18-24 hours before electroporaon, passage cells to aain an
opmal cell density at the me of electroporaon (i.e. 70 - 90% conuent for
most cell types).
For adherent cells: Plate cells at a density of 0.8 - 3.0 × 105 cells/ml
For suspension cells: Seed cells at a density of 1 - 2 × 106 cells/ml
2. Incubate cell cultures overnight.
B. Prepare Ingenio® Soluon/nucleic acid/cell mixture for electroporaon
(immediately before electroporaon)
1. Warm Ingenio® Electroporaon Soluon, trypsin-EDTA (if applicable) and
complete growth medium to room temperature.
2. Harvest and count cells to determine cell density (cells/ml).
3. Determine the total electroporaon volume required to perform the desired
number of electroporaons.
For 0.2 cm cuvees: Mulply number of electroporaons by 0.1 ml
For 0.4 cm cuvees: Mulply number of electroporaons by 0.25 ml
4. Calculate the cell volume required for all electroporaons:
For adherent cells: Use a nal cell density of 1 - 5 × 106 cells/ml
For suspension cells: Use a nal cell density of 10 × 106 cells/ml
5. Pipee the cell volume (from step B4) of harvested cells into a new tube and
centrifuge at 300 × g for 5 minutes. Aspirate the supernatant.
6. During centrifugaon, add pre-warmed complete culture medium to a new
culture dish to accept cells following electroporaon.
7. Aer centrifugaon, resuspend cells in Ingenio® Electroporaon Soluon us-
ing the volume determined in step B3.
(Cont. on next page)
Mirus Bio LLC
www.mirusbio.com │ [email protected] │ U.S. Toll Free: 844.MIRUSBIO (844.647.8724)│ Direct: +1.608.441.2852
Final Cell Density (cells/ml) × Volume from Step B3 (ml)
Harvested Cell Density from Step B2 (cells/ml)
Cell Volume (ml) =

Page 13
Ingenio® EZporator® Electroporaon System
Product User Manual | Instrucons for MIR 51000
EZporator® System Protocols (Connued)
8. Add the desired amount of nucleic acid to the Ingenio® Electroporaon Solu-
on and cell mixture. Mix gently but thoroughly. Do not create air bubbles.
For DNA: Use 20 µg DNA per 1 ml of cells as a starng point.
For siRNA: Use 250 nM siRNA (nal concentraon) as a starng point.
NOTE: For further opmizaon, refer to the Ingenio® Full Protocol.
C. Perform Electroporaon
1. Aliquot Ingenio® Soluon/nucleic acid/cell mixture into cuvees for electro-
poraon.
For 0.2 cm cuvees: Pipet 100 μl total mixture per cuvee
For 0.4 cm cuvees: Pipet 250 µl total mixture per cuvee
2. Electroporate the cells at room temperature.
NOTE: The opmal pulse condions or program sengs will vary depending
on the cell type and electroporator used. For most mammalian and insect cell
types, opmal condions fall within the following ranges in LV mode:
For 0.2 cm cuvees: 80-160 V (voltage) and 800-1000 µF (capacitance)
For 0.4 cm cuvees: 200-300 V (voltage) and 800-1000 µF (capacitance)
3. Immediately transfer the electroporated cells into the prepared culture dish
(from step B6). NOTE: The opmal post-electroporaon cell culture density
will depend on the cell type, transfected nucleic acid and post-electroporaon
incubaon period.
4. Incubate the electroporated cells in appropriate culture medium for 12-72
hours or as required. A culture medium change may be necessary for longer
incubaons.
5. Harvest cells and assay as required.
Mirus Bio LLC
www.mirusbio.com │ [email protected] │ U.S. Toll Free: 844.MIRUSBIO (844.647.8724)│ Direct: +1.608.441.2852

Page 14
Ingenio® EZporator® Electroporaon System
Product User Manual | Instrucons for MIR 51000
Opmizaon
Electroporaon parameters (e.g. cell density, nucleic acid amount, voltage) should
be opmized for each cell type to ensure best possible transfecon eciency and
cell viability. The suggesons below yield high eciency electroporaon of most
mammalian and insect cell types.
Cell Density and Passage Number
• Cell division. Passage cultures approximately 18-24 hours before electro-
poraon to ensure that cells are acvely dividing and reach the appropriate
density (i.e. 70-90% conuent for adherent cells or 2 - 4 × 106 cells/ml for sus-
pension cells) at the me of harvesng for electroporaon. In general, cells
should be in mid-logarithmic growth for opmal electroporaon.
• Cell density at electroporaon. Determine the opmal cell density for each cell
type to maximize electroporaon eciency. The opmal cell density for elec-
troporaon is typically between 1 - 10 × 106 cells/ml. Higher cell densies are
typically recommended for suspension cells (e.g. 1 x 107 cells/ml), whereas the
recommended range for adherent cells is 1 - 5 × 106 cells/ml.
• Cell passage number. Use of very low or very high passage cells may aect
experimental results. Use cells of similar passage number for experimental
reproducibility.
Nucleic Acid Purity and Concentraon
• Plasmid DNA. Use highly puried, sterile and contaminant-free DNA for elec-
troporaon. Plasmid DNA preparaons that are endotoxin-free and have
A260/280 absorbance rao of 1.8-2.0 are desirable. DO NOT use DNA prepared
using miniprep kits or DNA that is less than 0.5 mg/ml in concentraon. To
determine the best plasmid DNA concentraon for electroporaon, try DNA
concentraons in the range of 5-50 μg/ml of nal electroporaon volume.
• siRNA. Use siRNA that is highly pure, sterile and the correct sequence. Try siR-
NA in the range of 250-750 nM (nal concentraon in cuvee) to determine
the best siRNA concentraon for electroporaon.
Mirus Bio LLC
www.mirusbio.com │ [email protected] │ U.S. Toll Free: 844.MIRUSBIO (844.647.8724)│ Direct: +1.608.441.2852

Page 15
Ingenio® EZporator® Electroporaon System
Product User Manual | Instrucons for MIR 51000
Opmizaon
Pulse Condions
• Voltage. For cells resuspended in Ingenio® Electroporaon Soluon, the expo-
nenal decay pulse condions for most cell types fall within a voltage range of
80-160 V when using 0.2 cm cuvees (100 µl volume) and 200-300 V when
using 0.4 cm cuvees (250 µl volume). To opmize electroporaons, vary the
voltage in 10 V increments (starng at 200 V for 0.4 cm cuvees and 80 V for
0.2 cm cuvees).
• Time Constant. Time constant (TC) values will vary depending on electro-
poraon buer conducvity, volume in the cuvee, cell density and tempera-
ture. Typical TC values with Ingenio® Electroporaon Soluon range from 19-23
ms in 0.2 cm cuvees (100 µl volume) and 29-32 ms in 0.4 cm cuvees (250 µl
volume).
Example Voltage Titraon: BJ Fibroblasts were electroporated with a GFP-encoding
plasmid DNA at the specied voltages in 0.2 cm cuvees (100 µl volume). Delivery
eciency and cell viability were determined by ow cytometry 48 hours post-
electroporaon:
Mirus Bio LLC
www.mirusbio.com │ [email protected] │ U.S. Toll Free: 844.MIRUSBIO (844.647.8724)│ Direct: +1.608.441.2852
110 V 130 V 150 V 170 V

Page 16
Ingenio® EZporator® Electroporaon System
Product User Manual | Instrucons for MIR 51000
Pulse Condions for Common Cell Types
Mirus Bio LLC
www.mirusbio.com │ [email protected] │ U.S. Toll Free: 844.MIRUSBIO (844.647.8724)│ Direct: +1.608.441.2852
Cell Type Cuvee
Size (cm) Cell Density (×106 cells/ml) DNA (µg) Electroporaon Volume (µl) Voltage (V)
Primary Human
Keranocyte 0.2 0.4 22 5100 250 150 220
Primary MEF 0.2
0.4 52 5100 250 150 230
Primary Rat
Corcal Neuron 0.2 0.4 12 - 100 - 120 -
A549 0.2
0.4 5- 5- 250 - 280
BHK21 0.2
0.4 10 2 5100 250 150 280
CHO-K1 0.2
0.4 52 5100 250 150 280
COS-70.2
0.4 52 5100 250 150 260
HEK 293 0.2
0.4 52 5100 250 160 250
HEK 293T 0.2
0.4 5- 5- 250 - 250
HeLa 0.2
0.4 32 5100 250 130 260
Hepa 0.2
0.4 52 - 100 - 160 -
HepG2 0.2
0.4 52 5100 250 170 250
HL-60 0.2
0.4 10 2 5100 250 150 276
HUVEC 0.2
0.4 3- 5- 250 - 250
Jurkat 0.2
0.4 10 2 5100 250 150 260
K562 0.2
0.4 10 2 5100 250 130 250
MCF-70.2
0.4 32 - 100 - 150 -
NIH-3T3 0.2
0.4 10 2 5100 250 160 260
NIKS 0.2
0.4 22 5100 250 170 280
PC-12 0.2
0.4 32 5100 250 130 240
RAW 264.7 0.2
0.4 52 5100 250 150 260
SH-SY5Y 0.2
0.4 5- 5- 250 - 250
SK-BR-30.2
0.4 52 5100 250 160 260
SK-N-MC 0.2
0.4 52 5100 250 90 240
THP-10.2
0.4 10 2 5100 250 140 250
U-937 0.2
0.4 10 - 5- 250 - 260
Vero 0.2
0.4 52 - 100 - 170 -
Other
(Starng Point) 0.2 0.4 5-10 2 5100 250 80-160 200-300

Page 17
Ingenio® EZporator® Electroporaon System
Product User Manual | Instrucons for MIR 51000
Instrument Troubleshoong
Instrument does not power up: Verify that the power cord is fully inserted in the
instrument and wall outlet. To verify that the fuse is not blown, rst disconnect the
power cord from the instrument and then remove the fuse holder. See Maintenance
secon (page 20) for fuse replacement instrucons.
Error messages appear: The EZporator® System is constantly monitoring the param-
eters of some of its internal circuitry. In the case of a failed pulse, one of the follow-
ing messages will appear on the display:
SCR failure: The EZporator® has not detected an output pulse during the pulsing
operaon. A short in the electrode could be the cause. Unplug the generator for
90 seconds then reset by cycling the power and pulsing one more me. If the
same message reappears, contact Mirus Technical Support.
Charge failure: The EZporator® has detected a failure to charge its capacitors
properly. Press the ‘Pulse’ buon to reset this message. A very low line voltage
or a brownout may be the cause. Verify that the outlet powering the EZporator®
is adequately rated. If the message persists, contact Mirus Technical Support.
Setpoint too low: The user has entered a setpoint that is outside of normal
operang specicaons. Increase the voltage seng.
NOTE: Although the instrument rmware allows input voltages down to 2 V in LV
mode and 10 V in HV mode, these are below the recommended voltage sengs.
"Setpoint too low" or "SCR failure" error messages are likely to occur in this very low
voltage range because of the extended me needed to discharge capacitor banks
down to < 20 V. At ≥ 20 V, the instrument will reliably discharge and send pulses.
Arc discharge: Arcing is a complete or paral electrical discharge circumvenng the
sample and is oen accompanied by a spark-like ash and sound. Arcing may be
caused by a faulty cuvee or inappropriate voltage setpoint for the soluon used or
volume in the cuvee. Ensure that cells were properly resuspended in Ingenio®
Electroporaon Soluon and that nucleic acids are highly pure. Consider reducing
the voltage setpoint or increasing sample volume unl arcing is no longer a problem.
NOTE: There is no error message for arcing: however, the electroporated cell popu-
laon is unlikely to be viable should arcing occur.
Mirus Bio LLC
www.mirusbio.com │ [email protected] │ U.S. Toll Free: 844.MIRUSBIO (844.647.8724)│ Direct: +1.608.441.2852

Page 18
Ingenio® EZporator® Electroporaon System
Product User Manual | Instrucons for MIR 51000
Experimental Troubleshoong: Low Electroporaon Eciency
Mirus Bio LLC
www.mirusbio.com │ [email protected] │ U.S. Toll Free: 844.MIRUSBIO (844.647.8724)│ Direct: +1.608.441.2852
Problem Soluon
Cell density not
opmal at me of
electroporaon
Determine the best cell density for each cell type to maximize electro-
poraon eciency. For most suspension cells, a cell density of 1 × 107
cells/ml is recommended for electroporaon. For adherent cells, a range
of 1 - 5 × 106 cells/ml is recommended. Use of higher or lower densies
may increase cell viability depending on cell type.
Cells not acvely
dividing at the me
of electroporaon
Passage cells at least 18–24 hours before electroporaon to ensure that
the cells are acvely dividing and reach opmal cell density at the me of
electroporaon.
Subopmal DNA
concentraon
Conrm DNA concentraon and purity. Use plasmid DNA preps that have
an A260/280 absorbance rao of 1.8-2.0.
The opmal DNA concentraon generally ranges between 5-50 µg/ml of
nal electroporaon volume. Start with 20 µg/ml of total electroporaon
volume.
Low quality plasmid
DNA
Use highly puried, sterile, endotoxin and contaminant-free DNA for
transfecon. Do not use DNA prepared using miniprep kits as it might
contain high levels of endotoxin.
Incorrect vector
sequence
If you do not observe expression of your target insert, verify the se-
quence of the plasmid DNA.
Proper experimental
controls were not
included for plasmid
delivery
To verify ecient electroporaon, deliver a posive control such as a
luciferase or green uorescent protein (GFP) encoding plasmid.
To assess delivery eciency of plasmid DNA, use Mirus Bio Label IT®
Tracker™ Intracellular Nucleic Acid Localizaon Kit to label the target
plasmid or use Mirus Bio prelabeled Label IT® Plasmid Delivery Controls.
Subopmal siRNA
concentraon
The opmal siRNA concentraon generally ranges between 250-750 nM
nal concentraon. Use 250 nM siRNA as a starng point.
Incorrect siRNA
sequence
Ensure that the sequence of the siRNA is correct for the gene of interest.
More than one sequence may need to be tested for opmal knockdown
eciency and to ensure proper targeng.
Poor quality of siRNA Avoid siRNA degradaon by using RNase-free handling procedures and
plasc ware. Degradaon of siRNA can be detected on acrylamide gels.
Proper controls were
not included for
siRNA delivery
Recommended controls include: (1) Cells alone, (2) Serum-free medium +
Ingenio® Electroporaon Soluon + a non-targeng siRNA.
To verify ecient transfecon and knockdown, deliver a siRNA targeted
against a ubiquitous gene, e.g. GAPDH or Lamin A/C, followed by target
western blong or mRNA quancaon.
Electroporaon
incubaon me
Determine the opmal electroporaon incubaon me for each cell type
and experiment. Test a range of incubaon mes (e.g. 12-72 hours). The
best incubaon me is generally 24-48 hours.

Page 19
Ingenio® EZporator® Electroporaon System
Product User Manual | Instrucons for MIR 51000
Experimental Troubleshoong: High Cellular Toxicity
Mirus Bio LLC
www.mirusbio.com │ [email protected] │ U.S. Toll Free: 844.MIRUSBIO (844.647.8724)│ Direct: +1.608.441.2852
Problem Soluon
Voltage setpoint too
high for cell type Decrease the voltage by increments of 10 V unl viability improves.
Cells not transferred
immediately to cul-
ture vessel contain-
ing complete growth
medium
Transfer the cells from each cuvee to a culture dish containing warm
complete culture medium immediately aer each electroporaon.
Endotoxin-
contaminated plas-
mid DNA
Use highly puried, sterile, endotoxin and contaminant-free DNA for
electroporaon.
We recommend using Mirus Bio MiraCLEAN® Endotoxin Removal Kit (MIR
5900) for removal of any traces of endotoxin from your DNA preparaon.
Alternavely, use cesium chloride gradient or anion exchange puried
DNA which contains levels of endotoxin that do not harm most cells.
DO NOT use DNA prepared using miniprep kits as it might contain high
levels of endotoxin.
DNA preparaon has
too much salt If DNA was prepared using an ion exchange column with a nal ethanol precipitaon step, we recommend exchanging the DNA soluon to a salt-
free or low salt soluon, e.g. elute the DNA in water and add 5 mM NaCl.
Excessive amounts of
DNA in the nal
electroporaon mix
Reduce the amount of DNA used for electroporaon. DNA concentraons
as low as 5 µg/ml of the nal electroporaon volume can be used.
Compare toxicity levels against a cells + Ingenio® Electroporaon Soluon
control to assess the eects of the DNA transfected. If you sll see toxici-
ty, it is likely due to the cell mixture being too concentrated or presence
of too many lysed cells.
Expressed target
gene is toxic to cells Compare toxicity levels against a cells-alone control and cells electro- porated with an empty vector to assess the cytotoxic eects of the target
protein being expressed.
If a lower level of target gene expression is desired in your electro-
poraon experiments, consider reducing the amount of target plasmid. If
necessary, maintain the opmal DNA concentraon (20 µg) by using
carrier DNA such as an empty cloning vector.
Knockdown of an
essenal gene
If the electroporated siRNA is directed against a gene that is essenal to
the cell, cytotoxicity may be observed due to knockdown of the target
gene. Include a control with non-targeng siRNA to compare the cytotox-
ic eects of the gene being knocked down.
Cell morphology has
changed
Mycoplasma contaminaon can alter cell morphology and aect electro-
poraon eciency. Check your cells for Mycoplasma contaminaon. Use
a fresh frozen stock of cells or use appropriate anbiocs to eliminate
Mycoplasma.
A high or low cell passage number can make cells more sensive and
refractory to electroporaon. Maintain a similar passage number be-
tween experiments to ensure reproducibility.

Page 20
Ingenio® EZporator® Electroporaon System
Product User Manual | Instrucons for MIR 51000
Maintenance
The Ingenio® EZporator® Electroporaon System does not need to be calibrated and
requires no special maintenance beyond occasional cleaning. To clean, rst power
o the EZporator® Pulse Generator module and remove the power cord. Carefully
clean exterior surfaces using a lint-free cloth and avoid scratching the LCD display
window. Dampen (but do not soak) a so cloth with an aqueous soluon of 70-80%
isopropyl alcohol or a mild detergent for more thorough cleaning. Do not expose the
cuvee chamber to excess moisture.
Fuse Replacement
The EZporator® fuse is located at the back of the instrument, oset below the green
Power Buon (see below). Blown fuses can easily be replaced by following these
instrucons:
1. Power o the Pulse Generator and disconnect the power cord from the module be-
fore servicing the fuse.
2. Locate the fuse (see below) and insert a small at-blade screwdriver into the slot on
the exterior poron of the fuse cap. Push in and turn le to unlock and remove the
fuse cap and fuse.
3. Remove the blown fuse from the cap and replace with a new fuse. NOTE: Use only 5
mm x 20 mm, 2.5 A, 250 V type T (me delay) fuses.
4. Return the fuse and cap to the Pulse Generator module. Carefully turn right with the
screwdriver to ghten.
Mirus Bio LLC
www.mirusbio.com │ [email protected] │ U.S. Toll Free: 844.MIRUSBIO (844.647.8724)│ Direct: +1.608.441.2852
Fuse
Power Buon
AC Input