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Thermo Fisher Scientific
Neon Transfection System User Guide
user guide for Thermo Fisher Scientific models including: Neon Transfection System, MPK5000, MPK1025, MPK1096, MPK10025, MPK10096, Thermo Fisher Scientific, Invitrogen, electroporation, transfection, mammalian cells, primary cells, stem cells, nucleic acids, siRNA
Neon Transfection System User Guide (Pub. No. MAN0001557 B.0) Thermo Fisher Scientific (1 March 2021) Neon Transfection System - Thermo Fisher Scientific B.0 1 March 2021 Update for RoHS2 compliance and SKU list A.0 11 July 2014 New document Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all applicable Limited Use Label Licenses.
Neon Transfection System - Thermo Fisher 2. Remove the plastic bag containing the manual, the Neon ™ Pipette box containing the pipette, and then remove the plastic bag containing the power cords from the box. 3. Remove the Neon ™ device and the Neon pipette station from the box and place them on a flat, level surface. 4. Set up the Neon ™ Transfection System as described on ...
neon device man NeonTM Transfection System
USER GUIDE For transfecting mammalian cells, including primary and stem cells, with high transfection efficiency
Catalog Numbers MPK5000, MPK1025, MPK1096, MPK10025, MPK10096
Document Part Number 251055 Publication Number MAN0001557 Revision B.0
For Research Use Only. Not for use in diagnostic procedures.
Manufacturer: Life Technologies Corporation | 5781 Van Allen Way | Carlsbad, California 92008 USA
The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Revision history: Pub. No. MAN0001557
Revision B.0 A.0
Date 1 March 2021 11 July 2014
Description Update for RoHS2 compliance and SKU list New document
Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all applicable Limited Use Label Licenses. TRADEMARKS: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
©2021 Thermo Fisher Scientific Inc. All rights reserved.
Contents
CHAPTER 1 Product information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Upon receiving the device . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Unpacking instructions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Product contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 NeonTM transfection system contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 NeonTM kit contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
System components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 NeonTM device . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 NeonTM pipette station . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 NeonTM Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
System overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Description of parts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
NeonTM device . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 NeonTM pipette . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 NeonTM pipette station . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 NeonTM tube . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 NeonTM tips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
CHAPTER 2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Getting started . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Install the NeonTM device with pipette station . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Register the device . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Electroporation protocol options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Input values limit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Input window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Database window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Optimization window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Upgrade the firmware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
General guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Recommended kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Recommended buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 DNA quality and amount . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
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siRNA quality and amount . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Using the NeonTM Transfection System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Materials needed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Set up the NeonTM pipette station . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Prepare adherent cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Prepare suspension cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Electroporation protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Cleaning and maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Optimization protocol for DNA and siRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Materials needed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 General guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 24-well optimization protocol for adherent and suspension cell lines--day one . . . . 34 18-well optimization protocol for primary suspension blood cells--day one . . . . . . . 36 Optimization protocol--day two . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Optional: optimization protocol--day three . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
APPENDIX A Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 NeonTM device error messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
APPENDIX B Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Repackaging the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 Repackaging and storage instructions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Replace the Pipette Gripper . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
APPENDIX C Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Product specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
APPENDIX D Related products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Accessory products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 Additional products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 Cell culture media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 siRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
APPENDIX E Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Safety information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 Informational symbols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 Informations de sécurité . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Informational symbols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
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Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
APPENDIX F Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
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1
Product information
Product description
The NeonTM Transfection System is a novel, benchtop electroporation device that employs an electroporation technology by using the pipette tip as an electroporation chamber to efficiently transfect mammalian cells including primary and immortalized hematopoietic cells, stem cells, and primary cells.
The NeonTM Transfection System efficiently delivers nucleic acids, proteins, and siRNA into all mammalian cell types including primary and stem cells with a high cell survival rate. The transfection is performed using as few as 1 × 104 or as many as 5 × 106 cells per reaction using a sample volume of 10 µL or 100 µL in a variety of cell culture formats (60 mm, 6-well, 48-well, and 24-well).
The NeonTM Transfection System uses a single transfection kit (NeonTM Kit) that is compatible with various mammalian cell types including primary and stem cells thereby avoiding the need to determine an optimal buffer for each cell type.
The NeonTM Transfection System offers open and transparent protocols that are optimized for ease of use and simplicity. The NeonTM device is preprogrammed with one 24-well optimization protocol to optimize conditions for your nucleic acid/siRNA and cell type, or you can program and store up to 50 cell-specific protocols in the NeonTM device database. Optimized protocols for many commonly used cell types are also available at https://www.thermofisher.com/us/en/home/life-science/cell-culture/ transfection/neon-transfection-system/neon-transfection-system-cell-line-data.html to maximize transfection efficiencies for your cell types.
See "Description of parts" on page 12 for details on various parts of the system.
Features
Important features of the NeonTM Transfection System are listed below: · User-friendly NeonTM device benchtop design that easily fits in your tissue culture hood for easy, efficient transfection of a wide variety of mammalian cells including primary and stem cells · Ability to transfect 1 × 1045 × 106 cells per reaction in a sample volume of 10 µL or 100 µL in a variety of cell culture formats (60 mm, 6-well, 48-well, and 24-well) · Utilizes a single buffer system for all cell types except primary suspension blood cells · Simple touch screen interface for easy programming of electroporation parameters · Available with one pre-programmed 24-well optimization protocol and the option to customize up to 50 cell specific protocols · Built-in safety features in the device to enhance user safety
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NeonTM Transfection System User Guide
1 Chapter 1 Product information Upon receiving the device
Upon receiving the device
Examine the unit carefully for any damage incurred during transit. Any damage claims must be filed with the carrier. The warranty does not cover in-transit damage. To register the device, activate your warranty, and be notified of important updates, go to thermofisher.com.
Unpacking instructions
Consult the following instructions to unpack the NeonTM Transfection System. The weight of the NeonTM device is 13.2 pounds (6 kg).
1. Cut the plastic straps and remove the outer box. Save the outer box and other packaging material (in case you need to transport or ship the unit).
2. Remove the plastic bag containing the manual, the NeonTM Pipette box containing the pipette, and then remove the plastic bag containing the power cords from the box.
3. Remove the NeonTM device and the NeonTM pipette station from the box and place them on a flat, level surface.
4. Set up the NeonTM Transfection System as described on page 15.
Product contents
NeonTM transfection system contents
The contents of the NeonTM Transfection Systems are listed in the following table. The NeonTM Transfection System is shipped at room temperature.
See page 12 for specifications and description of the NeonTM Transfection System, and page 15 to set up the device.
Product NeonTM Transfection Device Specific Power Cord (for US/Canada/Taiwan/Japan, Europe, and UK) NeonTM Pipette NeonTM Pipette Station User Guide USB Memory Device
Quantity 1 4
1 1 1 1
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1 Chapter 1 Product information Product contents
NeonTM kit contents
The NeonTM Kits are used with the NeonTM Transfection Systems for efficient transfection of mammalian cells and are available as standalone products (see "Accessory products" on page 52). The kits consist of two components which are not sold individually (a Tips/Tubes Kit, and a Buffer Kit), and are available in two formats (for electroporation of 10 µL samples, and 100 µL samples).
NeonTM Kit components are listed in the following table, and are shipped at room temperature.
After receiving the kit, store buffers at 4 and tips/tubes at room temperature.
NeonTM Kit, 10 µL
NeonTM Kit, 100 µL
Item
Tips/Tubes Kit NeonTM Tips NeonTM Tubes Buffer Kit
Cat. No. MPK1025 (50 reactions) MPK1025K
25 tips (10 µL)
5 MPK1025B
Cat. No. MPK1096 (192 reactions) MPK1096K
96 tips (10 µL)
20 MPK1096B
Cat. No. MPK10025 (50 reactions) MPK10025K
25 tips (100 µL)
5 MPK10025B
Cat. No. MPK10096 (192 reactions) MPK10096K
96 tips (100 µL)
20 MPK10096B
Resuspension Buffer R (Proprietary)
1 mL
3 × 1 mL
10 mL
30 mL
Resuspension Buffer T (Proprietary)
1 mL
3 × 1 mL
10 mL
30 mL
Electrolytic Buffer E (Proprietary)
75 mL
2 × 150 mL
--
--
Electrolytic Buffer E2 (Proprietary)
--
--
75 mL
2 × 150 mL
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Chapter 1 Product information System components
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System components
NeonTM device
The NeonTM device is a simple, user friendly benchtop electroporation device. It is used with the NeonTM Pipette Station and NeonTM Kits to efficiently transfect mammalian cells including primary and stem cells. See "Description of parts" on page 12 for details.
Front view
1
1 Touchscreen
Rear view
3
45
2
1 6
1 USB port panel for USB memory device (unscrew the panel to access the port)
2 High voltage port (connect to the high voltage connector of the NeonTM Pipette Station)
3 Sensor port (connect to the sensor connector of the NeonTM Pipette Station)
4 Power switch
5 AC inlet (connect to the power cord, and plug into the power outlet on the wall)
6 Fan
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1 Chapter 1 Product information System components User interface 1 2
1 Digital Display shows the protocol in use and various protocol parameters
2 Touchscreen buttons to operate the device
NeonTM pipette station
The NeonTM Pipette Station is a unique component of the system that holds the NeonTM Pipette during electroporation, and protects the user from any electrical shock exposures. A high voltage and sensor connector which connects the pipette station to the NeonTM device. See "Description of parts" on page 12 for details.
2 3
1
1 Connector cable 2 Sensor connector
3 High voltage connector
NeonTM Kits
The NeonTM Kits (not supplied with the device) contain the NeonTM Tips, NeonTM Tubes, and buffers for electroporation. The NeonTM Kits are available in two formats for electroporation of 10 µL or 100 µL samples (See page 52 for ordering information). See page 12 for details on NeonTM Tips and Tubes.
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1 Chapter 1 Product information System overview
System overview
Unlike standard cuvette based electroporation, the NeonTM Transfection System uses a unique electroporation reaction chamber, the NeonTM Tip that delivers a high electric field to the biological sample. The NeonTM Tip maximizes the gap size between the two electrodes while minimizing the surface area of each electrode. As a result, the sample experiences a more uniform electric field, minimal pH change, less ion formation, and negligible heat generation. This next generation electroporation technology overcomes many of the limitations associated with standard cuvette based electroporation thereby increasing transfection efficiency and cell viability, and providing an ergonomic workflow.
The transfection occurs in the uniquely designed NeonTM Tip using simple 3-step procedure. 1. Load a mixture of harvested cells and molecules to be delivered (e.g., DNA, RNA, siRNA) into the NeonTM Tip. 2. Plug the NeonTM Pipette with NeonTM Tip into position in the NeonTM Pipette Station with NeonTM Tube; select your protocol on the device, and press Start. 3. Unplug the NeonTM Pipette and transfer your transfected cells into a tissue culture vessel containing the appropriate medium.
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1 Chapter 1 Product information Description of parts
Description of parts
NeonTM device
The NeonTM Device employs the pipette tip as an electroporation chamber to efficiently transfect mammalian cells including primary and immortalized hematopoietic cells, stem cells, and primary cells. The device is preprogrammed with a 24-well optimization protocol and supports a database to store up to 50 user-specified protocols. See page 9 for a front and rear view of the device.
NeonTM pipette
The NeonTM Pipette utilizes a positive displacement pipette mechanism for pipetting mixtures containing cells and nucleic acid or siRNA. The NeonTM Pipette is a fixed volume pipette and permanently calibrated at the manufacturing stage and does not require any further calibration. The NeonTM Pipette is designed for use with NeonTM Tips only. Do not use any other tips with the NeonTM Pipette.
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1 Chapter 1 Product information Description of parts
NeonTM pipette station
The NeonTM Pipette Station holds a NeonTM Pipette during electroporation procedures. The NeonTM Pipette Station is equipped with many safety sensors and protection mechanisms that protect the user from any exposures to an electrical shock. The NeonTM Pipette Station is connected to the NeonTM device using the high voltage and sensor connector (see page 15 for details). The NeonTM Pipette Station also holds the NeonTM Tube which has an electrode near the bottom that transfers the electric field from the electrode inside the NeonTM Tip.
1 2
3
1 Connector cable 2 Area to insert the NeonTM Tube
3 NeonTM Pipette Station
NeonTM tube
The NeonTM Tube holds the Electrolytic Buffer during electroporation and is inserted into the NeonTM Pipette Station. The NeonTM Pipette with the NeonTM Tip is then inserted into the NeonTM Tube which has an electrode near the bottom that transfers the electric field from the electrode inside the NeonTM Tip. The NeonTM Tubes are supplied with NeonTM Kits as well as available separately (see page 52).
To avoid contamination, we strongly recommend using the tubes for a maximum of 10 times only. We recommend changing tube and buffer when switching to a different plasmid DNA/siRNA or cell type.
Tube Specifications:
Material: Polystyrene
Capacity: 2.54 mL
2 1
1 Electrode 2 Buffer
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1 Chapter 1 Product information Description of parts
NeonTM tips
The NeonTM Tips are disposable tips composed of a tip and piston used with the NeonTM Pipette. The NeonTM Tips contain a gold-plated electrode to create a disposable electric chamber for the delivery of a high electric field to biological samples. The NeonTM Tips are supplied with NeonTM Kits in two formats to support operating volumes of 10 µL and 100 µL, respectively (see page 52 for ordering information). To ensure repeatability and eliminate variation of the transfection conditions within or between experiments, we recommend that you do not use the NeonTM Tip for more than 2 times. Oxide formation at the piston surface area can be generated if the tips are used more than 2 times, which decreases electrode function of the piston. Tip specifications: Material: Polypropylene Capacity: 10 µL or 100 µL
1 2
3
4 1 Mounting stem 2 Piston 3 Gold electrode 4 Tip
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Methods
Getting started
Install the NeonTM device with pipette station
1. Unpack the NeonTM device as instructed in "Unpacking instructions" on page 7.
2. Four power cords are shipped with the device to ensure that the cord you use is compatible with your local socket format.
3. Place the NeonTM device on a level laboratory bench. Keep the area around the unit clear to ensure proper ventilation of the unit.
Note: The NeonTM device has a small footprint and can be easily set up in the tissue culture hood for convenience.
4. For your safety: Position the device properly such that the power switch and AC inlet located on the rear of the unit (see page 9) are easily accessible. Be sure to position the device such that it is easy to disconnect the unit.
Note: Since NeonTM device is air-cooled, its surface may become hot during operation. When installing the device, leave a space of more than 10 cm from the back of the device.
5. Place the NeonTM Pipette Station near the NeonTM device.
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2 Chapter 2 Methods Getting started 6. Connect the high voltage and sensor connector on the NeonTM Pipette Station to high voltage port and sensor port on the rear side of NeonTM device, respectively.
Be sure to align the ridge indicated by a white arrow on the sensor connector on the NeonTM Pipette Station with a groove indicated by a white dot on the sensor port of the NeonTM device (see figure for details).
IMPORTANT! To connect or disconnect the sensor connector to the NeonTM device, always handle the sensor connector using the cord plug and not the cord cable.
7. Ensure the AC power switch is in the Off position ( see page 9).
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8. Attach the power cord to the AC inlet on the rear of the NeonTM device and then to the electrical outlet. Use only properly grounded AC outlets and power cords.
9. To turn on the power, press the main power switch on the rear of the unit to ON position. The digital display shows start up screen (see page 17).
10. The NeonTM device is operated by the touch screen on the front of the device. You can easily input electroporation parameters by lightly touching the touch screen with a fingertip or a touch screen pen. See 17 for details.
You are ready to use the NeonTM Transfection System. See page 25 for details.
Register the device
Visit thermofisher.com to register the device and activate your warranty or extended warranty, and ensure that you receive product updates, special offers, and faster service.
Electroporation protocol options
There are three options to select an electroporation protocol for your cell type: · If you already have the electroporation parameters for your cell type, input the parameters in the Input Window (see page 17). · If you wish to add cell-specific electroporation parameters to the database on the device for future use, input the parameters in the Database Window (see page 19). You can also view our library of protocols for commonly used cell types from thermofisher.com and in put the parameters in the Database Window (see below) for various cell types. · If you do not have any specific electroporation parameters for your cell type and wish to perform optimization, use the Optimization Window (see page 21).
Input values limit
The NeonTM device is designed to only input certain values and limits for each value are listed below. If your input value exceeds the maximum value, an error is displayed. Input Voltage range: 5002,500 V Input Pulse Width range: 1100 ms Input Pulse Number range: 110
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Input window
To create a cell specific protocol, if you already have the electroporation parameters for your cell type:
1. Press the power switch (located at the rear of the unit, see page 9) to turn ON the NeonTM device. The unit checks to ensure that the NeonTM Pipette Station is connected to the device and then the start up screen is displayed.
2. Press Voltage to activate the number key pad to input voltage value. Press the desired voltage value and press Done to save the value.
Note: If any input value is out of the limit, an error message is displayed and the lowest value of limit is automatically set.
3. Press Width to activate the number key pad to input width value. Press the desired width value and press Done to save the value.
4. Press Pulses to activate the number key pad to input pulse value. Press the desired pulse value and press Done to save the value.
5. If you wish to save these electroporation parameters, press Save on the main screen to save the protocol in the database.
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6. Press the desired protocol number button to edit the protocol. The selected protocol is highlighted. 7. Once the Edit screen is displayed, enter the User name by pressing the key pad buttons. The
cursor automatically moves to the next field Protocol and is highlighted red. Continue to enter the information for Voltage, Width, and Pulse. 8. Press Enter to save the information in the database. 9. Proceed to preparing cells (see pages 2728) and DNA, and setting up the NeonTM Pipette Station for electroporation (see page 25).
Database window
Enter cell-specific protocols into the database. The database can store up to 50 cell-specific protocols. 1. Press the power switch (located at the rear of the unit, see page 9) to turn ON the NeonTM device. The unit checks to ensure that the NeonTM Pipette Station is connected to the device and then the start up screen is displayed.
2. Press Database button to start the database window. To scroll through the protocols in the database, use the right/left scroll buttons near the Database button.
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The Database window shows: · Number button: Indicates protocol number · User and Protocol: Displays the user and protocol name · Parameters (Voltage, Width, Pulse): Displays the electroporation parameter for each protocol · Function buttons (Load, Edit, and Delete): Used to load, edit, or delete a protocol. The function buttons are activated only after a protocol is selected. · Page scroll: To scroll to or
3. Press the desired protocol number button to edit the protocol. The selected protocol is highlighted.
4. Once the Edit screen is displayed, enter the User name by pressing the key pad buttons. The cursor automatically moves to the next field Protocol and is highlighted red.
Continue to enter the information for Voltage, Width, and Pulse.
If you wish to password protect the protocol, enter the Password (up to 7 characters) and Repeat Password information using the key pad.
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5. Press Enter to save the information in the database. To exit the edit screen without saving the parameters, press X.
6. The database window is displayed. Press the desired protocol and then press Load to load electroporation parameters from the database.
7. Proceed to preparing cells (see pages 2728) and DNA, and setting up the NeonTM Pipette Station for electroporation (see page 25).
8. To delete a protocol from the database, select the protocol by pressing the protocol number button. Press Delete. If the protocol in the database was password protected, a password screen is displayed. Enter the password and press Enter to delete the protocol.
Optimization window
Perform optimization of electroporation parameters using the preprogrammed 24-well optimization protocol. These protocols are locked and cannot be edited.
1. Press the power switch (located at the rear of the unit, see page 9) to turn ON the NeonTM device. The unit checks to ensure that the NeonTM Pipette Station is connected to the device and then the start up screen is displayed.
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2. Press Optimization button to start the optimization window. To scroll through the protocols, use the right/left scroll buttons near the Optimization button. The Optimization window shows: · Number button: Indicates protocol number · User and Protocol: Displays the optimization and well number · Parameters (Voltage, Width, Pulse): Displays the electroporation parameter for each protocol · Load Function buttons: Used to load a protocol. The Load button is activated only after a protocol is selected. · Page scroll: To scroll to or
3. Press the desired protocol number button. The selected protocol is highlighted. Press Load to load the protocol. To exit the screen without loading the protocol, press X.
4. The electroporation parameters are displayed on the start up screen.
5. Proceed to preparing cells (see pages 2728) and DNA, and setting up the NeonTM Pipette Station for electroporation (see page 25).
Upgrade the firmware
Upgrades for the NeonTM device firmware are available. To download NeonTM device firmware upgrades, go to thermofisher.com. Follow instructions on the page to download the upgrades.
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General guidelines
Recommended kits
To use the NeonTM device for electroporation of mammalian cells, you need to purchase the NeonTM Kits. Ordering information is on page 52. Do not use any other kits with the unit.
Note: To obtain the best results, follow these recommendations:
· Based on your initial results, you may need to optimize the electroporation parameters for your cell
type and DNA/siRNA. A preprogrammed 24-well optimization protocol is included in the device for your convenience.
· Before using the device with your samples, ensure that you are able to insert and use the NeonTM
Pipette and Tip correctly into the NeonTM Pipette Station (see page 25 for details).
· Wear gloves, laboratory coat, and safety glasses during electroporation. · Always use the NeonTM device with NeonTM Kits for electroporation of mammalian cells. · The NeonTM Transfection System is compatible for use with most mammalian cells including primary
and stem cells.
· Use high quality DNA and siRNA to obtain good transfection efficiency. · Follow the guidelines on pages 2728 for cell preparation. · Use an appropriate GFP (green fluorescent protein) construct or siRNA control (see page 24 for
details) to determine transfection efficiency.
· Discard the NeonTM Tips after 2 usages and NeonTM Tubes after 10 usages as a biological hazard. We
strongly recommend changing tube and buffer when switching to a different plasmid DNA/siRNA or cell type.
· Visit thermofisher.com for a library of electroporation protocols for commonly used cell types.
Recommended buffers
The NeonTM Kits contain two Resuspension Buffers. Use the appropriate Resuspension Buffer based on the voltage.
Resuspension Buffer R:
Use Resuspension Buffer R for all cell types and electroporation protocols. For high voltage protocols (1900V), optimize with both Resuspension Buffer R and T. If arcing occurs with Resuspension Buffer R consider switching to Resuspension Buffer T.
Resuspension Buffer T:
Use Resuspension Buffer T with high voltage protocols of 1900V or more. Cell-specific NeonTM transfection protocols available at https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/neontransfection-system/neon-transfection-system-cell-line-data.html indicate the type of Resuspension buffer for use with each cell type.
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DNA quality and amount
The quality and concentration of DNA used for electroporation plays an important role for the transfection efficiency. We strongly recommend using high quality plasmid purification kits such as PureLinkTM HiPure Plasmid DNA Purification Kits (see page 52) to prepare DNA.
· Resuspend the purified DNA in deionized water or TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) at a concentration between 15 µg/µL. Concentrations may vary depending on cell type.
· The DNA amount should not exceed 10% of total volume used. · Check the purity of the purified DNA preparation by measurement of the A260/280 ratio. The ratio
should be at least 1.8 for electroporation. · The device has been routinely tested with 47 kb plasmids and plasmids up to approximately
20 kb should not be a problem. Using plasmids larger than 20 kb will most likely lower transfection efficiency.
IMPORTANT! Do not precipitate DNA with ethanol to concentrate DNA. Concentrated DNA by ethanol precipitation shows poor transfection efficiency and cell viability due to salt contamination.
siRNA quality and amount
The quality and concentration of siRNA used for electroporation plays an important role for the transfection efficiency. We strongly recommend using high quality siRNA such as StealthTM, SilencerTM Select, or SilencerTM siRNA.
· The recommended starting siRNA concentration is 100250 µM in nuclease-free water. · The siRNA amount should not exceed 10% of total volume used.
Controls
GFP control
To initially assess transfection efficiency for your cell type using fluorescent microscopy, we recommend using a plasmid encoding GFP (green fluorescent protein) or any colored variant of GFP (ClontechTM or equivalent). For best results, the vector encoding the GFP should have the following features:
· Strong promoter active in a variety of mammalian cells such as the immediate early CMV (cytomegalovirus) promoter
· SV40 polyadenylation signals downstream of the GFP gene for proper processing of the 3' end of the GFP mRNA.
· Antibiotic selection marker · pUC origin of replication for propagation in E. coli
siRNA control
For siRNA experiments, use BLOCK-iTTM Fluorescent OligoTM for electroporation or SilencerTM Select GAPDH Positive Control siRNA (see page 52) to assess transfection efficiency.
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Using the NeonTM Transfection System
Instructions are provided in this section to use the NeonTM device with the NeonTM Pipette Station and NeonTM Kits for electroporation of mammalian cells. General instructions to prepare cells for use with the NeonTM Transfection System are described below. For primary and stem cell types, use the established methods developed in the laboratory.
See "Optimization protocol for DNA and siRNA" on page 33 if you wish to use the preprogrammed optimization protocol.
Materials needed
See page 52 for ordering information. · Cells · NeonTM Kits · High quality DNA at a concentration of 15 µg/µL in deionized water or TE buffer, or high quality RNAi duplex at a concentration of 100250 µM in nuclease-free water (see page 24) · Cell culture plates containing the appropriate medium · D-PBS or Phosphate buffered saline (PBS) without Ca2+ and Mg2+ (see page 52) · Trypsin/EDTA or TrypLETM Express (Cat. No. 12563) for adherent cells · CountessTM Automated Cell Counter (see page 52) or equivalent
Note: If you are a first time user of the NeonTM Transfection System, we recommend that you review the protocol below and ensure that you are able to insert and use the NeonTM Pipette and Tip correctly into the NeonTM Pipette Station (see below for details) before you start using the system with your samples.
IMPORTANT!
· To obtain the highest transfection efficiency and low non-specific effects, optimize transfection
conditions by varying electrical parameters as described in "Optimization protocol for DNA and siRNA" on page 33 using the pre-programmed optimization protocol in a 24-well format.
· Since the cell culture conditions vary from user to user, be sure to use low passage number, actively
dividing cells (for dividing cells)
· For siRNA transfection, the concentration of RNAi duplex required will vary depending on the efficacy
of the duplex. After the initial results, vary the siRNA final concentration from 10200 nM.
Note: The siRNA concentration in the NeonTM transfection protocol refers to the siRNA concentration in the culture medium and not to the siRNA concentration in the electroporation mix in the NeonTM Tip.
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Set up the NeonTM pipette station
1. Ensure the NeonTM Pipette Station is connected to the NeonTM device (see page 15). 2. Fill the NeonTM Tube with 3 mL of Electrolytic Buffer (use Buffer E for 10 µL NeonTM Tip and Buffer
E2 for 100 µL NeonTM Tip).
Note: Make sure that the electrode on the side of the tube is completely immersed in buffer.
3. Insert the NeonTM Tube into the NeonTM Pipette Station until you hear a click sound.
Note: Make sure that the side electrode of the NeonTM tube is well connected to the side ball plunger of the NeonTM Pipette Station (see figure on the left below for correct position).
4. The station is ready for use. Proceed to "Prepare adherent cells" on page 27.
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Prepare adherent cells
1. Cultivate the required number of cells (7090% confluent on the day of transfection) by seeding a flask containing fresh growth medium 12 days prior to electroporation. For most optimized protocols, seed with: · 5 × 104 to 2 × 105 cells for each 10 µL NeonTM Tip · 5 × 105 to 2 × 106 cells for each 100 µL NeonTM Tip
2. Pre-warm an aliquot of culture medium containing serum, PBS (without Ca2+ and Mg2+), and Trypsin/EDTA solution to 37.
3. Aspirate the media from cells and rinse the cells using PBS (without Ca2+ and Mg2+).
4. Trypsinize the cells using Trypsin/EDTA or TrypLETM Express (Cat. no. 12563).
5. After neutralization, harvest the cells in growth medium with serum (0.75 mL for a 10 µL NeonTM Tip or 7.5 mL for a 100 µL NeonTM Tip).
6. Take an aliquot of trypsinized cell suspension and count cells to determine the cell density.
7. Transfer the cells to a 1.5 mL microcentrifuge tube or a 15 mL conical tube and centrifuge the cells at 100400 × g for 5 minutes at room temperature.
8. Wash cells with PBS (without Ca2+ and Mg2+) by centrifugation at 100400 × g for 5 minutes at room temperature.
9. Aspirate the PBS and resuspend the cell pellet in Resuspension Buffer R (or Resuspension Buffer T for programs 1900V) at a final density of 1.0 × 107 cells/mL. Gently pipette the cells to obtain a single cell suspension.
Note: Avoid storing the cell suspension for more than 1530 minutes at room temperature, which reduces cell viability and transfection efficiency. The resuspension cell density may be adjusted to accommodate the recommended cell numbers for the electroporation protocol (see page 29) or optimization protocols (see pages 3440).
10. Prepare 24-well plates by filling the wells with 0.5 mL of culture medium containing serum and supplements without antibiotics and pre-incubate plates in a humidified 37/5% CO2 incubator. If you are using other plate format, see page 29 for plating medium volume recommendations.
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Prepare suspension cells
1. Cultivate the required number of cells (cell density 13 × 106 cells/T-25 flask) by seeding a flask containing fresh growth medium 12 days prior to electroporation. For most optimized protocols, seed with: · 15 × 105 cells for each 10 µL NeonTM Tip · 15 × 106 cells for each 100 µL NeonTM Tip
2. Pre-warm an aliquot (500 µL per sample for 10 µL NeonTM Tips or 5 mL for 100 µL NeonTM Tips) of culture medium containing serum. Also prepare an appropriate volume of PBS (without Ca2+ and Mg2+).
3. Take an aliquot of cell culture and count the cells to determine the cell density.
4. Transfer the cells to a microcentrifuge tube or 15 mL conical tube and pellet the cells by centrifugation at 100400 × g for 5 minutes at room temperature.
5. Wash the cells with PBS (without Ca2+ and Mg2+) and pellet the cells by centrifugation at 100 400 × g for 5 minutes at room temperature.
6. Aspirate the PBS and resuspend the cell pellet in Resuspension Buffer R (or Resuspension Buffer T for programs 1900V) at a final density of 2.0 × 107 cells/mL. Gently pipette the cells to obtain a single cell suspension.
Note: Avoid storing the cell suspension for more than 1530 minutes at room temperature, which reduces cell viability and transfection efficiency. The resuspension cell density maybe adjusted to accommodate the recommended cell numbers for the electroporation protocol (see page 29) or optimization protocols (see pages 3440).
7. Prepare 24-well plates by filling the wells with 0.5 mL of culture medium containing serum and supplements without antibiotics and pre-incubate plates in a humidified 37/5% CO2 incubator. If you are using other plate format, see page 29 for plating medium volume recommendations.
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Electroporation protocol
1. Make sure you have appropriate number of cells prepared as described on pages 2728, have the plasmid DNA or siRNA at the suggested concentrations (see page 24), and prepare a plate containing culture medium without antibiotics to transfer the electroporated cells.
For details on optimizing the transfection efficiency of your cells, see "Optimization protocol for DNA and siRNA" on page 33.
2. For each electroporation sample, the recommended amount of plasmid DNA or siRNA, cell number, and volume of plating medium per well are listed below. Use Resuspension Buffer T for primary suspension blood cells.
Format Cell Type
96-well 48-well
Adherent Suspension
Adherent Suspension
24-well 12-well 6-well
Adherent Suspension
Adherent Suspension
Adherent
Suspension
60 mm 10 cm
Adherent Suspension
Adherent Suspension
DNA (µg)
0.250.5 0.51 0.251 0.52
0.52 0.53 0.53 0.53 0.53 (10 µL) 530 (100 µL) 0.53 (10 µL) 530 (100 µL) 530 530 530 530
siRNA (nM)
NeonTM Tip
Vol. plating medium
Cell no.
Buffer R or Buffer T[1]
10200
10 µL
100 µL 12 × 104 10 µL/well
10 µL
25 × 104 10 µL/well
10-200
10 µL
250 µL 2.55 × 104 10 µL/well
10 µL
512.5 × 104
10 µL/well
10-200
10 µL
500 µL 0.51 × 105 10 µL/well
10 µL
12.5 × 105 10 µL/well
10-200
10 µL
1 mL
12 × 105 10 µL/well
10 µL
25 × 105 10 µL/well
10-200 10 µL/100 µ L
2 mL
24 × 105 10 µL or 100 µL/well
10 µL/100 µ L
0.41 × 106 10 µL or 100 µL/well
10-200 10-200
100 µL 100 µL 100 µL 100 µL
5 mL 10 mL
0.51 × 106 100 µL/well 12.5 × 106 100 µL/well 12 × 106 100 µL/well 25 × 106 100 µL/well
[1] Use Resuspension Buffer T for primary suspension blood cells.
3. Set up a NeonTM Tube with 3 mL Electrolytic Buffer (use Buffer E for 10 µL NeonTM Tip and Buffer E2 for 100 µL NeonTM Tip) into the NeonTM Pipette Station (see page 26).
4. Set the desired pulse conditions on the device based on your cell type (see "Electroporation protocol options" on page 17).
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5. Transfer the appropriate amount of plasmid DNA/siRNA into a sterile, 1.5 mL microcentrifuge tube.
6. Add cells to the tube containing plasmid DNA/siRNA and gently mix. See the table for cell concentration, DNA, and plating volumes to use.
7. To insert a NeonTM Tip into the NeonTM Pipette, press the push-button on the pipette to the second stop to open the clamp.
8. Insert the top-head of the NeonTM Pipette into the NeonTM Tip until the clamp fully picks up the mounting stem of the piston (see below)
9. Gently release the push-button, continuing to apply a downward pressure on the pipette, ensuring that the tip is sealed onto the pipette without any gaps.
Note: Ensure that the NeonTM Pipette and Tip are tightly connected without a gap (see figure on the left) for trouble-free pipetting and proper electrical connection.
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10. Press the push-button on the NeonTM Pipette to the first stop and immerse the NeonTM Tip into the cell-DNA/siRNA mixture. Slowly release the push-button on the pipette to aspirate the cellDNA/siRNA mixture into the NeonTM Tip.
Note: Avoid air bubbles during pipetting as air bubbles cause arcing during electroporation leading to lowered or failed transfection. If you notice air bubbles in the tip, discard the sample and carefully aspirate the fresh sample into the tip again without any air bubbles.
11. Insert the NeonTM Pipette with the sample vertically into the NeonTM Tube placed in the NeonTM Pipette Station until you hear a click sound. Ensure that the pipette projection is inserted into the groove of the pipette station.
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Note: Ensure the metal head of the NeonTM Pipette is tightly connected to the ball plunger inside of the NeonTM Pipette Station and to the NeonTM Tube (see figure on the left for the correct position).
12. Ensure that you have selected the appropriate electroporation protocol and press Start on the touchscreen.
13. The NeonTM device automatically checks for the proper insertion of the NeonTM Tube and NeonTM Pipette before delivering the electric pulse.
Note: Monitor the NeonTM Tip during electroporation to see if there is any arcing (sparks) that is caused by the presence of bubbles in the tip. Arcing results in low transfection efficiency and cell viability.
14. After delivering the electric pulse, Complete is displayed on the touchscreen to indicate that electroporation is complete.
15. Slowly remove the NeonTM Pipette from the NeonTM Pipette Station and immediately transfer the samples from the NeonTM Tip by pressing the push-button on the pipette to the first stop into the prepared culture plate containing prewarmed medium.
Note: We strongly recommend loading electroporated cells into growth medium without antibiotics that can greatly reduce the viability of your cells after transfection.
16. To discard the NeonTM Tip, press push-button to the second stop into an appropriate biological hazardous waste container.
17. Repeat Steps 716 for the remaining samples. Be sure to change the NeonTM Tips after using it twice and NeonTM Tubes after 10 usages. Use a new NeonTM Tip and NeonTM Tube for each new plasmid DNA sample.
18. Gently rock the plate to assure even distribution of the cells. Incubate the plate at 37 in a humidified CO2 incubator.
19. If you are not using the NeonTM device, turn the power switch on the rear to OFF.
20. Assay samples to determine the transfection efficiency (e.g., fluorescence microscopy or functional assay) or gene knockdown (for siRNA).
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Optimization
Based on your initial results, you may need to optimize the electroporation parameters for your cell type. See "Optimization protocol for DNA and siRNA" on page 33 for using the 18-well or preprogrammed 24-well optimization protocol on the NeonTM device.
Cleaning and maintenance
Clean the surface of the NeonTM device and NeonTM Pipette Station with a damp cloth. Do not use harsh detergents or organic solvents to clean the unit. The NeonTM Pipette is permanently calibrated at the manufacturer and does not require any further calibration.
IMPORTANT! Avoid spilling any liquid inside of the NeonTM Pipette Station to prevent any build up of rust on the ball plunger in the pipette station.
In case you accidentally spill any liquid (e.g., buffer, water, coffee) inside the NeonTM Pipette Station, disconnect the station from the main device and wipe the station using dry laboratory paper. Invert and allow the station to completely dry for 24 hours at room temperature. Do not use the oven to dry the NeonTM Pipette Station. If the station does not work after drying, contact Technical Support. For any other repairs and service, contact Technical Support. Do not perform any repairs or service on the NeonTM device yourself as it is a high voltage hazard and to avoid any damage to the unit or voiding your warranty.
Optimization protocol for DNA and siRNA
Electroporation is mainly dependent on the combination of three electric parameters such as the electric field, pulse width, and pulse number. Based on your initial results, you may need to optimize the electroporation parameters for your cell type especially the hard-to-transfect cells. The NeonTM device is preprogrammed with a 24-well optimization protocol using the 10 µL or 100 µL NeonTM Tip that allows you to quickly optimize electric parameters for many adherent and suspension cell lines within days.
For primary blood suspension cells, use the 18-well optimization protocol with Resuspension Buffer T as described on page 36.
Materials needed
See page 52 for ordering information. · NeonTM 10 µL or 100 µL Kit · Cells in Resuspension Buffer (prepared as described on pages 2728) · High quality DNA at a concentration of 15 µg/µL in deionized water or TE buffer or high quality RNAi duplex at a concentration of 100250 µM in nuclease-free water (see page 24) · Cell culture plates containing the appropriate medium
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General guidelines
General guidelines for optimization are described below. For a detailed protocols, see page 34 for adherent and suspension cell line optimization, and page 36 for primary suspension blood cell optimization.
Optimization for plasmid
1. Perform 24-well optimization using the preprogrammed parameters.
2. Based on results from Step 1, perform optimization using narrower (bracket) parameters.
3. Based on results from Step 2, further refine the parameters to obtain optimal conditions (this is optional step).
Optimization for siRNA
1. Perform 24-well optimization using the preprogrammed parameters.
2. Based on results from Step 1, perform optimization using narrower (bracket) parameters.
3. Based on results from Step 2, perform optimization by varying siRNA final concentrations to 10 nM, 30 nM, 100 nM, and 200 nM.
24-well optimization protocol for adherent and suspension cell lines--day one
1. Make sure you have cells prepared as described on pages 2728, have the DNA or siRNA, and prepare a 24-well plate containing 0.5 mL culture medium with serum and without antibiotics to transfer the electroporated cells. Prepare enough cells and plasmid DNA/siRNA for at least 30 transfections.
2. For each electroporation sample using the 10 µL NeonTM Tip in 24-well format, see table. For using the 100 µL NeonTM Tip in 24-well format, adjust the amounts listed in the table appropriately by 10fold.
Cell type Adherent
Suspension
Cell no. 1 × 105/well
2 × 105/well
DNA
siRNA
0.5 µg DNA/well 15 µg/plate
1 µg DNA/well 30 µg/plate
50 pmol in 10 µL tip
100 nM per well
100 pmol in 10 µL tip
200 nM per well
Resuspension Buffer R 10 µL/well
285 µL/plate
10 µL/well 270 µL/plate
3. Set up a NeonTM Tube with 3 mL Electrolytic Buffer (use Buffer E for 10 µL NeonTM Tip and Buffer E2 for 100 µL NeonTM Tip) into the NeonTM Pipette Station containing the cell-DNA/siRNA mixture as described on page 26.
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4. Press Optimization and load the optimization protocols to begin electroporation using the parameters listed below.
Sample
1 2 3
Well no.
A1 A2 A3
Pulse voltage
Pulse width
Pulse no.
Results
Transfection efficiency
Cell viability
Use pre-optimized parameter or control without electroporation.
1400
20
1
1500
20
1
4
A4
1600
20
1
5
A5
1700
20
1
6
A6
1100
30
1
7
B1
1200
30
1
8
B2
1300
30
1
9
B3
1400
30
1
10
B4
1000
40
1
11
B5
1100
40
1
12
B6
1200
40
1
13
C1
1100
20
2
14
C2
1200
20
2
15
C3
1300
20
2
16
C4
1400
20
2
17
C5
850
30
2
18
C6
950
30
2
19
D1
1050
30
2
20
D2
1150
30
2
21
D3
1300
10
3
22
D4
1400
10
3
23
D5
1500
10
3
24
D6
1600
10
3
5. After electroporation, immediately remove the NeonTM Pipette and transfer samples from the 10 µL NeonTM Tip into prewarmed 0.5 mL culture medium. For 100 µL NeonTM Tip, dilute samples 10fold in 900 µL medium and transfer 100 µL of the sample to 0.4 mL prewarmed culture medium.
6. Repeat Steps 35 for the remaining samples.
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Chapter 2 Methods Optimization protocol for DNA and siRNA
7. Gently rock the plate to assure even distribution of the cells. Incubate the plate at 37 in a humidified CO2 incubator.
8. Assay samples to determine the transfection efficiency (e.g., fluorescence microscopy or functional assay) or gene knockdown (for siRNA). Select the best conditions and proceed to the next day's experiment, "Optimization protocol--day two" on page 38.
18-well optimization protocol for primary suspension blood cells--day one
1. Make sure you have cells prepared as described on pages 2728, have the DNA or siRNA, and prepare 18-wells of a 24-well plate containing 0.5 mL culture medium with serum and without antibiotics to transfer the electroporated cells. Prepare enough cells and plasmid DNA or siRNA for at least 20 transfections.
2. For each electroporation sample using the 10 µL NeonTM Tip in 18-wells of a 24-well plate, see table.
Cell type
Primary blood suspension cells
Cell no. 2 × 105/well
DNA
1 µg DNA/well 20 µg/plate
siRNA
100 pmol in 10 µL tip
200 nM per well
Resuspension Buffer T
10 µL/well 180 µL/plate
3. Set up a NeonTM Tube with 3 mL Electrolytic Buffer E into the NeonTM Pipette Station and NeonTM Tip containing the cell-DNA/siRNA mixture.
4. Input the electroporation parameters in the Input window and perform electroporation using the parameters listed below.
Sample
1 2 3 4 5 6 7 8 9 10 11 12
Well no.
A1 A2 A3 A4 A5 A6 B1 B2 B3 B4 B5 B6
Pulse voltage
Pulse width
Pulse no.
Results
Transfection efficiency
Cell viability
Use pre-optimized parameter or control without electroporation.
2000
20
1
2050
20
1
2100
20
1
2150
20
1
2200
20
1
2250
20
1
2300
20
1
2350
20
1
2400
15
1
2450
15
1
2500
15
1
36
NeonTM Transfection System User Guide
Chapter 2 Methods Optimization protocol for DNA and siRNA
2
(continued)
Sample
13 14 15 16 17 18
Well no.
C1 C2 C3 C4 C5 C6
Pulse voltage
2000 2050 2100 2150 2200 2250
Pulse width
15 15 15 15 15 15
Pulse no.
2 2 2 2 2 2
Results
Transfection efficiency
Cell viability
5. After electroporation, immediately remove the NeonTM Pipette and transfer samples from the 10 µL NeonTM Tip into prewarmed 0.5 mL culture medium.
6. Repeat Steps 35 for the remaining samples.
7. Gently rock the plate to assure even distribution of the cells. Incubate the plate at 37 in a humidified CO2 incubator.
8. Assay samples to determine the transfection efficiency (e.g., fluorescence microscopy or functional assay) or gene knockdown (for siRNA). Select the best conditions and proceed to the next day's experiment, "Optimization protocol--day two" on page 38.
NeonTM Transfection System User Guide
37
2
Chapter 2 Methods Optimization protocol for DNA and siRNA
Optimization protocol--day two
Select the best transfection conditions obtained from the previous experiment and fine-tune the optimization by narrowing the Pulse Voltage.
For example, if you obtained optimal conditions between 1,500 V, 20 ms and 1,400 V, 30 ms, (underlined in the table) perform optimization using these narrower parameters as below.
1. Make sure you have cells prepared as described on pages 2728, have the DNA or siRNA, and prepare 18- or 24-wells of a 24-wells plate with 0.5 mL culture medium with serum and without antibiotics to transfer the electroporated cells.
2. For each electroporation sample using the 10 µL NeonTM Tip, see table.
For using the 100 µL NeonTM Tip in 24-well format, adjust the amounts listed in the table appropriately by 10fold.
Cell type Adherent
Suspension
Primary Suspension Blood Cells
Format 24-well
24-well
18-well
Cell no. 1 × 105/well
DNA
0.5 µg DNA/well 15 µg/plate
2 × 105/well
1 µg DNA/well 30 µg/plate
12 × 105/well
0.51 µg DNA/well
20 µg/plate
siRNA
50 pmol in 10 µL tip 100 nM per
well
100 pmol in 10 µL tip
200 nM per well
100 pmol in 10 µL tip
200 nM per well
Resuspension Buffer Buffer R
10 µL/well 285 µL/plate
Buffer R 10 µL/well 270 µL/plate
Buffer R 10 µL/well 180 µL/plate
3. Set up a NeonTM Tube with 3 mL Electrolytic Buffer (use Buffer E for 10 µL NeonTM Tip and Buffer E2 for 100 µL NeonTM Tip) into the NeonTM Pipette Station and NeonTM Tip containing the cell-DNA/siRNA mixture.
4. Perform electroporation using the parameters listed on the table:
Sample
1 2 3 4 5 6
Well no.
A1 A2 A3 A4 A5 A5
Pulse voltage
1450 1475 1500 1525 1550 1575
Pulse width
20 20 20 20 20 20
Pulse no.
1 1 1 1 1 1
Results
Transfection efficiency
Cell viability
38
NeonTM Transfection System User Guide
Chapter 2 Methods Optimization protocol for DNA and siRNA
2
(continued)
Sample
7 8 9 10 11 12 13
Well no.
B1 B2 B3 B4 B5 B6 C1
Pulse voltage
Pulse width
Pulse no.
Results
Transfection efficiency
Cell viability
1375
30
1
1400
30
1
1425
30
1
1450
30
1
1475
30
1
1500
30
1
Control containing DNA but no electroporation pulse.
5. After electroporation, immediately remove the NeonTM Pipette and transfer the samples from the 10 µL NeonTM Tip into prewarmed 0.5 mL culture medium. For 100 µL NeonTM Tip, dilute samples 10fold in 900 µL medium and transfer 100 µL of the sample to 0.4 mL prewarmed culture medium.
6. Repeat Steps 35 for the remaining samples.
7. Gently rock the plate to assure even distribution of the cells. Incubate the plate at 37 in a humidified CO2 incubator.
8. Assay samples to determine the transfection efficiency (e.g., fluorescence microscopy or functional assay) or gene knockdown (for siRNA).
9. Select the best conditions and proceed to the next day's experiment, "Optional: optimization protocol--day three" on page 40.
NeonTM Transfection System User Guide
39
2
Chapter 2 Methods Optimization protocol for DNA and siRNA
Optional: optimization protocol--day three
For further optimization, repeat experiments by varying other conditions such as multiple pulsations. This is optional and depends on the cell type.
For siRNA, you can vary the amount of siRNA from 10200 nM.
1. Make sure you have cells prepared as described on pages 2728, have the DNA or siRNA, and prepare 18- or 24-wells of a 24-well plate containing 0.5 mL culture medium with serum and without antibiotics to transfer the electroporated cells.
2. For each electroporation sample using the 10 µL NeonTM Tip, see table.
For using the 100 µL NeonTM Tip in 24-well format, adjust the amounts listed in the table appropriately by 10fold.
Cell Type Adherent
Suspension
Primary Suspension Blood Cells
Format 24-well
24-well
18-well
Cell no. 1 × 105/well
DNA
0.5 µg DNA/well 15 µg/plate
2 × 105/well
1 µg DNA/well 30 µg/plate
12 × 105/well
0.51 µg DNA/well
20 µg/plate
siRNA
50 pmol in 10 µL tip 100 nM per
well
100 pmol in 10 µL tip
200 nM per well
100 pmol in 10 µL tip
200 nM per well
Resuspension Buffer Buffer R
10 µL/well 285 µL/plate
Buffer R 10 µL/well 270 µL/plate
Buffer R 10 µL/well 180 µL/plate
3. Set up a NeonTM Tube with 3 mL Electrolytic Buffer (use Buffer E for 10 µL NeonTM Tip and Buffer E2 for 100 µL NeonTM Tip) into the NeonTM Pipette Station and NeonTM Tip containing the cell-DNA/siRNA mixture.
4. Perform electroporation using the parameters listed in the table:
Sample
1 2 3 4 5 6 7
Well no.
A1 A2 A3 A4 A5 A6 B1
Pulse voltage
1450 1475 1500 1525 1550 1575 1375
Pulse width
10 10 10 10 10 10 10
Pulse no.
2 2 2 2 2 2 3
Results
Transfection efficiency
Cell viability
40
NeonTM Transfection System User Guide
Chapter 2 Methods Optimization protocol for DNA and siRNA
2
(continued)
Sample
8 9 10 11 12 13
Well no.
B2 B3 B4 B5 B6 C1
Pulse voltage
Pulse width
Pulse no.
Results
Transfection efficiency
Cell viability
1400
10
3
1425
10
3
1450
10
3
1475
10
3
1500
10
3
Control containing DNA but no electroporation pulse.
5. After electroporation, immediately remove the NeonTM Pipette and transfer the samples from the 10 µL NeonTM Tip into prewarmed 0.5 mL culture medium. For 100 µL NeonTM Tip, dilute samples 10fold in 900 µL medium and transfer 100 µL of the sample to 0.4 mL prewarmed culture medium.
6. Repeat Steps 35 for the remaining samples and incubate the plate.
7. Assay samples to determine the transfection efficiency (e.g., fluorescence microscopy or functional assay) or gene knockdown (for siRNA).
8. Select the best conditions and save these parameters into the database for your cell type.
NeonTM Transfection System User Guide
41
A
Troubleshooting
Troubleshooting
Problem No power (the display remains blank when the power is turned on) Connection error message displayed
Error messages
Cause AC power cord is not connected Pipette or tube is incorrectly inserted
The sensor connector is not connected
Solution
Check AC power cord connections at both ends. Use the correct cords.
· Properly insert the NeonTM Pipette into the NeonTM Pipette Station as described on page 29. The metal head of the pipette should be tightly connected to the ball plunger inside the pipette station.
· Properly insert the NeonTM Tube into the NeonTM Pipette Station as described on page 26. The side electrode on the tube should be tightly connected to the ball plunger inside the pipette station.
· Avoid spilling any liquid into the pipette station to prevent any build up of rust on the ball plunger in the pipette station.
· Be sure to connect the sensor connector of the NeonTM Pipette Station to the sensor port on the rear of the NeonTM device.
· Make sure the mark on the cable plug and the instrument connector is aligned correctly (see page 15)
--
See page 46 for a description of
error messages.
42
NeonTM Transfection System User Guide
(continued) Problem
Connection failure
If the error persists and all connections are correct Arcing (sparks) NeonTM Transfection System User Guide
A Appendix A Troubleshooting Troubleshooting
Cause
Solution
No NeonTM Tip is inserted or the NeonTM Tip is inserted incorrectly
Make sure that the NeonTM Tip is inserted into NeonTM Pipette correctly as described on page 29. There should be no gap between the tip and the top head of the pipette.
No buffer in the tube or no sample in the tip
Be sure to add 3 mL of the appropriate Electrolytic Buffer to NeonTM Tube. The electrode in the tube must be completely immersed in buffer.
Be sure to add sample in Resuspension Buffer to the NeonTM Tip.
Wrong buffers used
Use the Electrolytic Buffer (Buffer E for 10 µL tip and Buffer E2 for 100 µL tip) in the NeonTM Tube and the sample in Resuspension Buffer in the NeonTM Tip. Do not switch buffers or use any other buffer as these buffers are specifically designed for electroporation with the NeonTM device.
High voltage connector is not connected
Be sure to connect the high voltage connector of the NeonTM Pipette Station to the high voltage port on the rear of the NeonTM device (see page 15).
Perform self diagnostics test
Perform self diagnostics test by clicking on on the main screen. During the self diagnostics test, the device checks a variety of parameters and indicates if it is OK or there is a problem. If the self diagnostics is OK, ensure that all connections are correct as described in this section before contacting Technical Support.
Air bubbles in the NeonTM Tip
Avoid any air bubbles in the NeonTM Tip while aspirating the sample.
High voltage or pulse length settings
Reduce the voltage or pulse length settings.
43
A Appendix A Troubleshooting Troubleshooting (continued) Problem Arcing (sparks) Low cell survival rate
Low transfection efficiency
Cause
Solution
Accidentally used salt-precipitated Do not precipitate DNA with
DNA
ethanol to concentrate DNA as
it can cause arcing due to salt
contamination.
Poor DNA quality
Use high quality plasmid DNA for transfection (see page 24 for guidelines and recommendations on DNA quality).
Cells are stressed or damaged
Avoid severe conditions during cell harvesting especially high speed centrifugation and pipette cells gently.
Avoid using over confluent cells or cells at high densities as this may affect the cell survival after electroporation.
After electroporation, immediately plate the cells into prewarmed culture medium without antibiotics.
Multiple use of the same NeonTM Tip
Do not use the same NeonTM Tip for electroporation for more than 2 times because the repeated application of electric pulses reduce the tip quality and impairs their physical integrity.
Poor optimization of electrical parameters
Perform optimization for your cell type as described on page 33.
Poor plasmid DNA quality or the plasmid DNA is low
Use high quality plasmid DNA for transfection (see page 24 for guidelines and recommendations on DNA quality).
Start with 0.5 µg plasmid DNA per sample.
Incorrect cell density
Cell densities >3 × 105 or <5 × 104 per sample drastically reduces transfection efficiency. Use 5 × 1041.5 × 105 cells per 10 µL per sample.
44
NeonTM Transfection System User Guide
A Appendix A Troubleshooting Troubleshooting
(continued) Problem
Low transfection efficiency Non-reproducible transfection efficiency
High energy error
Cause Mycoplasma contaminated cells Inconsistent cell confluency or passage number Multiple use of NeonTM Tip and NeonTM Tube
Used high electrical parameters
Solution
Test cells for Mycoplasma contamination.
Start a new culture from a fresh stock.
Always use cells with low passage number and harvest cells with comparable confluency levels.
Do not use the same NeonTM Tip for more than 2 times because the repeated application of electric pulses reduce the tip quality and impairs their physical integrity.
Do not use the same NeonTM Tube for more than 10 times.
Always use a new NeonTM Tip and NeonTM Tube for different plasmid DNA samples to avoid any crosscontamination.
Set lower voltage or duration.
NeonTM Transfection System User Guide
45
A
Appendix A Troubleshooting NeonTM device error messages
NeonTM device error messages
This section describes the error messages displayed. Most of the error messages are self explanatory and after fixing the error, you should be able to continue with the protocol. Contact Technical Support if you need to send the device for servicing.
Error message Please connect station
Check tip for air bubbles.
Please enter user name Please enter protocol name Password incorrect, please re-enter Input voltage, pulse width, or pulse number error
Action
The NeonTM Pipette Station is not connected properly; ensure that the sensor connector is connected to the sensor port on the rear of the device (see page 15).
Remove the solution and aspirate the sample into the tip again without any air bubbles. Press OK to exit the screen.
All protocols in the database need a user name. Enter the user name and press OK to exit the screen.
All protocols in the database need a protocol name. Enter the user name and press OK to exit the screen.
Re-enter the 4-digit password and press OK to exit the screen.
The input voltage, pulse width, or pulse number is out of range. The valid range is displayed on the screen. Please enter the valid value and press OK to exit the screen.
46
NeonTM Transfection System User Guide
B
Maintenance
Repackaging the instrument
If you need to send the device to Thermo Fisher Scientific for warranty issues, or you wish to transport the instrument to another location, repackage the unit as follows.
Note: Prior to sending the device, ensure the device is properly decontaminated if the device is exposed to any viable biological agents, radioactive materials, or hazardous chemicals (toxic, carcinogenic, mutagenic, toxic for reproduction, sensitizing, and/or have not been fully tested). Contact Technical Support for a decontamination protocol and to obtain a Returns Goods Authorization (RGA) number and return shipping instructions.
Repackaging and storage instructions
1. Turn off the main power switch at the rear of the device and detach the power cord from the rear of device.
2. Disconnect the high voltage and sensor connector connected to the pipette station via the connector at the back of the unit.
3. Place the instrument in the original box including the original packing foam.
4. Tape the box securely and place appropriate shipping labels for shipping the instrument to InvitrogenTM. Always transport the box with the unit in the upright position.
5. If the device is not to be used for extended periods of time, store the repackaged device in an upright position at 4 to 40.
NeonTM Transfection System User Guide
47
B Appendix B Maintenance Replace the Pipette Gripper
Replace the Pipette Gripper
1. Insert the NeonTM Tube into the NeonTM Pipette Station followed by the NeonTM Pipette vertically into the NeonTM Tube until it clicks into place
2. Rotate the NeonTM Pipette counterclockwise; then, hold the SUS Head by hand to disassemble it completely.
48
NeonTM Transfection System User Guide
B Appendix B Maintenance
Replace the Pipette Gripper
3. Once the SUS Head is separated, check the internal parts in the following order:
1
2
3
4
1 Pipette body 2 Gripper
3 Spring 4 SUS Head
4. To replace Gripper, place the new gripper and spring in order into the Pipette body.
1
1 Gripper
5. Next, assemble the SUS Head first by hand and then completely by inserting the NeonTM Pipette vertically into the NeonTM Tube in the NeonTM Pipette Station until it clicks into place.
6. Lastly, hold the NeonTM Pipette and rotate the NeonTM Pipette clockwise to finish the assembly.
NeonTM Transfection System User Guide
49
B Appendix B Maintenance Replace the Pipette Gripper
7. When the SUS Head is assembled, the appearance of the pipette should be adjusted so that the and are collinear.
1
2
50
NeonTM Transfection System User Guide
C
Specifications
Product specifications
Operating Power:
Output: Pulse Width: Maximum Duty Cycle: Charging Time: Altitude: Operating Temperature: Maximum Relative Humidity: Degree of Protection: Protective Earthing: Installation Category: Instrument Type: Device Dimensions: Pipette Station Dimensions: Device Weight: Built-in Features:
100240 VAC, 2.1 A, 150 W, Frequency 50/60 Hz, 0.5-2.5 kV 1-100 ms 0.1
Maximum 8 seconds Up to 2,000 meters
5 to 40 Up to 80%
IPX0 Class I (earthed)
II Benchtop unit 9.2 inches (w) × 11.8 inches (l) × 8.66 inches (h) 5.91 inches (diameter); 5.51 inches (h) 13.2 pounds (6 kg) Touch screen (800 × 480 pixels), digital display
The NeonTM Transfection System including the NeonTM Pipette Station is compatible with standard nonhazardous laboratory reagents. Do not use organic solvents in the tip/tubes or with the device.
NeonTM Transfection System User Guide
51
D
Related products
Accessory products
Additional products
The following products are for use with the NeonTM Transfection System and are available separately. For more information, go to thermofisher.com or contact Technical Support.
NeonTM Kit, 10 µL
Product
NeonTM Kit, 100 µL
NeonTM Pipette NeonTM Pipette Station NeonTM Tubes
Dulbecco's Phosphate-Buffered Saline (D-PBS) (1X), liquid without Ca2+ and Mg2+ BLOCK-iTTM Fluorescent OligoTM for electroporation SilencerTM Select GAPDH Positive Control siRNA (human, mouse, rat) SilencerTM Select negative Control No. 1 siRNA SilencerTM Cy3TM labeled GAPDH siRNA (human, mouse, rat) SilencerTM FAMTM labeled GAPDH siRNA (human, mouse, rat) CountessTM Automated Cell Counter PureLinkTM HiPure Plasmid Miniprep Kit PureLinkTM HiPure Plasmid Midiprep Kit PureLinkTM HiPure Plasmid Filter Midiprep Kit PureLinkTM HiPure Plasmid Maxiprep Kit
Quantity 1 kit (50 reactions) 1 kit (192 reactions) 1 kit (50 reactions) 1 kit (192 reactions)
1 each 1 each 1 pack of 100 500 mL
75 µL 5 nmol
40 nmol 5 nmol
5 nmol
1 each 25 preps 25 preps 25 preps 25 preps
Catalog no. MPK1025 MPK1096 MPK10025 MPK10096 MPP100 MPS100 MPT100 14190-144
13750062 4390849
4390844 AM4649
AM4650
C10227 K2100-02 K2100-04 K2100-14 K2100-07
52
NeonTM Transfection System User Guide
D Appendix D Related products Accessory products
(continued) Product
PureLinkTM HiPure Plasmid Filter Maxiprep Kit MagMAXTM 96 Total RNA Isolation Kit TaqManTM Gene Expression Cells-to-CTTM Kit alamarBlueTM
Quantity 25 preps 96 reactions 100 reactions
25 mL
Catalog no. K2100-17 AM1830 AM1728 DAL1025
Cell culture media
A large variety of cell culture media and products for mammalian cells including primary and stem cells is available from InvitrogenTM. For more information, contact Technical Support.
siRNA
A large variety of siRNA products including StealthTM RNAi, SilencerTM Select RNAi,SilencerTM RNAi, or standard unmodified siRNA is available from InvitrogenTM. For more information, contact Technical Support.
NeonTM Transfection System User Guide
53
E
Safety
Safety information
Follow the instructions in this section to ensure safe operation of the NeonTM Transfection device. The NeonTM Transfection System is designed to meet EN61010-1 Safety Standards. To ensure safe, reliable operation, always operate theNeonTM Transfection System according to the instructions in this manual. Failure to comply with the instructions in this manual may create a potential safety hazard, and will void the manufacturer's warranty and void the EN61010-1 safety standard certification. Life Technologies is not responsible for any injury or damage caused by use of this instrument when operated for purposes which it is not intended. All repairs and service should be performed by Life Technologies.
· Always ensure that the power supply input voltage matches the voltage available in your location. · For operating environment, see page 51. · This device is air-cooled so its surfaces become hot during operation. When installing the device,
leave a space of more than 10 cm (4 inches) around it. · Never insert metallic objects into the air vents of the device as this could result in electrical shock,
personal injury and equipment damage. · Always set the main switch on the power supply unit to OFF before connecting the power cord to
the wall outlet. · Always ensure that the grounding terminal of the device and that of the wall outlet are properly
connected. Connect the power cord to a grounded, 3-conductor power outlet. · To avoid potential shock hazard, make sure that the power cord is properly grounded. · Be sure to position the instrument such that it is easy to disconnect the unit. · Be sure to set the main switch to OFF, unplug the power cord, and secure the pipette station before
moving the device.
Informational symbols
Symbol and description
CAUTION! Risk of danger. Consult the manual for further safety information.
CAUTION! Risk of electrical shock.
WEEE (Waste Electrical and Electronic Equipment) symbol indicates that this product should not be disposed of in unsorted municipal waste. Follow local municipal waste ordinances for proper disposal provisions to reduce the environmental impact of WEEE. This instrument meets European requirement WEEE Directive 2012/19/EU.
54
NeonTM Transfection System User Guide
Appendix E Safety Informations de sécurité
E
(continued) ON (power)
Symbol and description
OFF (power)
Protective earth (ground)
The CE mark symbolizes that the product conforms to all applicable European Community provisions for which this marking is required. Operation of the NeonTM Transfection System is subject to the conditions described in this manual. The protection provided by the device may be impaired if the instrument is used in a manner not specified by the manufacturer.
This product conforms to UL 61010-1, CAN/CSA C22.2 No.61010-1 "Safety Requirements for Electrical Equipment for Measurement, Control, and Laboratory Use, Part l: General Requirements." Instruments bearing the TUV symbol are certified by TUV Product Services to be in conformance with the applicable safety standard for the US and Canada.
Regulatory Compliance Mark indicates conformity with Australian standards for electromagnetic compatibility.
China RoHS EFUP 25
Informations de sécurité
Suivez les instructions de cette section pour vous assurer d'utiliser l'appareil NeonTM Transfection en toute sécurité. Le NeonTM Transfection System est conçu pour répondre aux normes de sécurité EN61010-1. Pour assurer un fonctionnement sûr et fiable, utilisez toujours le NeonTM Transfection System conformément aux instructions de ce manuel. Le non-respect des instructions contenues dans ce manuel pourrait engendrer un éventuel danger pour la sécurité et annulerait la garantie du fabricant ainsi que la certification à la norme de sécurité NF EN61010-1. Life Technologies ne peut être tenu responsable de toute blessure ou dommage provoqués par l'utilisation de cet instrument dans des buts autres que ceux prévus. Toutes les réparations et la maintenance doivent être effectuées par Life Technologies.
· Assurez-vous toujours que la tension d'entrée de l'alimentation corresponde à la tension disponible sur le lieu d'utilisation.
· Pour l'environnement d'exploitation, consultez la page 51.
· Cet appareil étant aéroréfrigéré, ses surfaces chauffent lorsqu'il fonctionne. Lors de l'installation de l'appareil, laissez un espace supérieur à 10 cm (4 pouces) autour de celui-ci.
· N'introduisez jamais d'objets métalliques dans les orifices d'aération de l'appareil, car cela pourrait provoquer un choc électrique, des blessures corporelles ou endommager l'équipement.
· Mettez toujours le commutateur principal de l'alimentation sur OFF (ARRÊT) avant de brancher le cordon d'alimentation sur la prise murale.
NeonTM Transfection System User Guide
55
E
Appendix E Safety Informational symbols
· Vérifiez toujours que la borne de mise à la terre de l'appareil et celle de la prise murale sont correctement raccordées. Branchez le cordon d'alimentation sur une prise d'alimentation à 3 conducteurs et reliée à la terre.
· Pour éviter tout risque potentiel de choc électrique, vérifiez que le cordon d'alimentation est correctement relié à la terre.
· Veillez à placer l'instrument de manière à pouvoir le débrancher facilement. · Veillez à mettre le commutateur principal sur OFF (ARRÊT), à débrancher le cordon d'alimentation
et à immobiliser la station à pipettes avant de déplacer l'appareil.
Informational symbols
Symbol and description
MISE EN GARDE ! Risque de danger. Consulter le manuel pour d'autres renseignements de sécurité.
MISE EN GARDE ! Risque de choc électrique.
Le symbole DEEE (Déchets d'équipements électriques et électroniques) indique que ce produit ne doit pas être mis au rebut avec des déchets ménagers non triés. Suivez la réglementation locale relative à l'élimination des déchets usuels pour réduire l'impact environnemental des DEEE. Rendez-vous sur www.invitrogen.com/weee pour prendre connaissance des options de collecte et de recyclage..
ON (MARCHE) (alimentation)
OFF (ARRÊT) (alimentation)
Protection par la mise à la terre (masse)
La marque CE est un symbole indiquant que le produit est conforme à toutes les dispositions applicables de la Communauté européenne pour lesquelles ce marquage est obligatoire. L'utilisation du NeonTM Transfection System est soumise aux conditions décrites dans ce manuel. Si vous utilisez l'instrument d'une manière non spécifiée par le fabricant, la protection offerte par l'appareil pourrait s'en trouver détériorée.
Ce produit est conforme à UL 61010-1, CAN/CSA C22.2 No.61010-1 «Exigences de sécurité pour l'équipement électrique pour la mesure, le contrôle et l'utilisation en laboratoire, Partie l : Généralité Les exigences.» Les instruments portant le symbole TUV sont certifiés par TUV Product Services conforme à la norme de sécurité applicable aux États-Unis et au Canada.
La marque de conformité réglementaire indique qu'elle est conforme aux normes australiennes compatibilité électromagnétique
56
NeonTM Transfection System User Guide
(continued)
Chine RoHS EFUP 25
Symbol and description
Appendix E Safety Chemical safety
E
Chemical safety
WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards, ensure laboratory personnel read and practice the general safety guidelines for chemical usage, storage, and waste provided below. Consult the relevant SDS for specific precautions and instructions:
· Read and understand the Safety Data Sheets (SDSs) provided by the chemical manufacturer
before you store, handle, or work with any chemicals or hazardous materials. To obtain SDSs, see the "Documentation and Support" section in this document.
· Minimize contact with chemicals. Wear appropriate personal protective equipment when handling
chemicals (for example, safety glasses, gloves, or protective clothing).
· Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with
sufficient ventilation (for example, fume hood).
· Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturer
cleanup procedures as recommended in the SDS.
· Handle chemical wastes in a fume hood. · Ensure use of primary and secondary waste containers. (A primary waste container holds the
immediate waste. A secondary container contains spills or leaks from the primary container. Both containers must be compatible with the waste material and meet federal, state, and local requirements for container storage.)
· After emptying a waste container, seal it with the cap provided. · Characterize (by analysis if needed) the waste generated by the particular applications, reagents,
and substrates used in your laboratory.
· Ensure that the waste is stored, transferred, transported, and disposed of according to all local,
state/provincial, and/or national regulations.
· IMPORTANT! Radioactive or biohazardous materials may require special handling, and disposal
limitations may apply.
WARNING! HAZARDOUS WASTE (from instruments). Waste produced by the instrument is potentially hazardous. Follow the guidelines noted in the preceding General Chemical Handling warning.
WARNING! 4L Reagent and Waste Bottle Safety. Four-liter reagent and waste bottles can crack and leak. Each 4-liter bottle should be secured in a low-density polyethylene safety container with the cover fastened and the handles locked in the upright position.
NeonTM Transfection System User Guide
57
E
Appendix E Safety Biological hazard safety
Biological hazard safety
WARNING! Potential Biohazard. Depending on the samples used on this instrument, the surface may be considered a biohazard. Use appropriate decontamination methods when working with biohazards.
WARNING! BIOHAZARD. Biological samples such as tissues, body fluids, infectious agents, and blood of humans and other animals have the potential to transmit infectious diseases. Conduct all work in properly equipped facilities with the appropriate safety equipment (for example, physical containment devices). Safety equipment can also include items for personal protection, such as gloves, coats, gowns, shoe covers, boots, respirators, face shields, safety glasses, or goggles. Individuals should be trained according to applicable regulatory and company/ institution requirements before working with potentially biohazardous materials. Follow all applicable local, state/provincial, and/or national regulations. The following references provide general guidelines when handling biological samples in laboratory environment.
· U.S. Department of Health and Human Services, Biosafety in Microbiological and Biomedical
Laboratories (BMBL), 5th Edition, HHS Publication No. (CDC) 21-1112, Revised December 2009; found at:
https://www.cdc.gov/labs/pdf/CDC-BiosafetymicrobiologicalBiomedicalLaboratories-2009P.pdf
· World Health Organization, Laboratory Biosafety Manual, 3rd Edition,
WHO/CDS/CSR/LYO/2004.11; found at:
www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf
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Documentation and support
Customer and technical support
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Note: For SDSs for reagents and chemicals from other manufacturers, contact the manufacturer.
Limited product warranty
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1 March 2021
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