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User Manual

Neon

™

Transfection System

For transfecting mammalian cells, including primary and
stem cells, with high transfection efficiency

Catalog no. MPK5000

Rev. Date: 1 April 2010

Manual Part no. 25-1055

MAN0001557

Summary of Contents for invitrogen Neon

Page 1: ...Manual Neon Transfection System For transfecting mammalian cells including primary and stem cells with high transfection efficiency Catalog no MPK5000 Rev Date 1 April 2010 Manual Part no 25 1055 MAN0001557 ...

Page 2: ...ii ...

Page 3: ...Parts 3 Methods 6 Getting Started 6 General Guidelines 12 Using the Neon Transfection System 14 Optimization Protocol for DNA and siRNA 22 Troubleshooting 31 Neon Device Error Messages 33 Appendix 34 Repackaging the Instrument 34 Product Specifications 35 Safety Information 36 Accessory Products 38 Technical Support 39 Purchaser Notification 40 iii ...

Page 4: ...iv ...

Page 5: ... transfection of mammalian cells and are available separately from Invitrogen page 38 The kits are available in two formats for electroporation of 10 μL and 100 μL samples The following components are included with the Neon Kit The Neon Kits are shipped at room temperature Upon receipt store the kit at room temperature After use store buffers at 4ºC and all remaining kit components at room tempera...

Page 6: ...ve the outer box and other packaging material in case you need to transport or ship the unit 2 Remove the plastic bag from the top 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 Neon pipette station from the box and place on a flat level surface 4 Set up the Neon Transfection Sys...

Page 7: ...w The USB port need to unscrew the panel to view the port is used to connect a USB memory drive The AC inlet is to connect to the power outlet on the wall and high voltage and sensor port is to connect the high voltage and sensor connector of the Neon Pipette Station to the unit Sensor port AC inlet Continued on next page High voltage port Power switch USB port panel Fan vii ...

Page 8: ...ip and Neon Tube is then used with the Neon Pipette Station for electroporation of mammalian cells The Neon Pipette Station contains two electrodes Connector User Interface The touchscreen user interface of the Neon device consists of The touchscreen buttons to operate the device The Digital Display that shows the protocol that is in use and various parameters of the protocol Digital display Touch...

Page 9: ... cell specific protocols in the Neon device database Optimized protocols for many commonly used cell types are also available on www invitrogen com neon for your convenience to maximize transfection efficiencies for your cell types See page 3 for details on various parts of the system System Components The Neon Transfection System consists of Neon Device The Neon device is a simple user friendly b...

Page 10: ...NA into the Neon Tip 2 Plug the Neon Pipette with Neon Tip into position in the Neon Pipette Station with Neon Tube select your protocol on the device and press Start 3 Unplug the Neon Pipette and transfer your transfected cells into a tissue culture vessel containing the appropriate medium Features Important features of the Neon Transfection System are listed below User friendly Neon device bench...

Page 11: ... a database to store up to 50 user specified protocols See page vii for a front and rear view of the device Neon Pipette The Neon Pipette utilizes a positive displacement pipette mechanism for pipetting mixtures containing cells and nucleic acid or siRNA The Neon Pipette is a fixed volume pipette and permanently calibrated at the manufacturing stage and does not require any further calibration The...

Page 12: ...c field from the electrode inside the Neon Tip Area to insert Neon Tube Neon Pipette Station Connector Neon Tube The Neon Tube holds the Electrolytic Buffer during electroporation and is inserted into the Neon Pipette Station The Neon Pipette with the Neon Tip is then inserted into the Neon Tube which has an electrode near the bottom that transfers the electric field from the electrode inside the ...

Page 13: ...rmats to support operating volumes of 10 μL and 100 μL respectively page 38 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 Neon 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 electro...

Page 14: ... that it is easy to disconnect the unit Note Since Neon 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 Neon Pipette Station near the Neon device 6 Connect the high voltage and sensor connector on the Neon Pipette Station to high voltage port and sensor port on the rear side of Neon...

Page 15: ...ecial 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 below If you wish to add cell specific electroporation parameters to the database on the device for future use input the parameters in the ...

Page 16: ...r 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 6 Press the desired protocol number button to edit the...

Page 17: ...roll through the protocols in the database use the right left scroll buttons near the Database button 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 ...

Page 18: ... 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 pages 16 17 and DNA and setting up the Neon Pipette Station for electroporation page 14 8 To delete a protocol from the database select ...

Page 19: ... 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 next or previous page 3 Press the desired protocol number button The selected protocol is highlighted Press Load to load the p...

Page 20: ... 16 17 for cell preparation Use an appropriate GFP green fluorescent protein construct or siRNA control see next page for details to determine transfection efficiency Discard the Neon Tips after 2 usages and Neon 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 www invitrogen com neon for ...

Page 21: ...ency 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 Stealth Silencer Select or Silencer siRNA The recommended starting siRNA concentration is 100 250 μM in nuclease free water The siRNA amount should no...

Page 22: ...r equivalent R E C O M MEND A T I O N If you are a first time user of the Neon Transfection System we recommend that you review the protocol below and ensure that you are able to insert and use the Neon Pipette and Tip correctly into the Neon 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...

Page 23: ... Make sure that the electrode on the side of the tube is completely immersed in buffer 3 Insert the Neon Tube into the Neon Pipette Station until you hear a click sound Note Make sure that the side electrode of the Neon tube is well connected to the side ball plunger of the Neon Pipette Station see figure on the left below for correct position O Ball plunger Side electrode ⅹ O Ball plunger Side el...

Page 24: ...ells to determine the cell density 8 Transfer the cells to a 1 5 mL microcentrifuge tube or a 15 mL conical tube and centrifuge the cells at 100 400 g for 5 minutes at room temperature 9 Wash cells with PBS without Ca2 and Mg2 by centrifugation at 100 400 g for 5 minutes at room temperature 10 Aspirate the PBS and resuspend the cell pellet in Resuspension Buffer R at a final density of 1 0 107 cel...

Page 25: ...nd pellet the cells by centrifugation at 100 400 g for 5 minutes at room temperature 6 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 7 Aspirate the PBS and resuspend the cell pellet in Resuspension Buffer R or Resuspension Buffer T at a final density of 2 0 107 cells mL Gently pipette the cells to obtain a single c...

Page 26: ... 10 μL 500 μL 1 2 5 105 10 μL well Adherent 0 5 3 10 μL 1 2 105 10 μL well 12 well Suspension 0 5 3 10 200 10 μL 1 mL 2 5 105 10 μL well Adherent 0 5 3 10 μL 5 30 100 μL 10 μL 100 μL 2 4 105 10 μL or 100 μL well 6 well Suspension 0 5 3 10 μL 5 30 100 μL 10 200 10 μL 100 μL 2 mL 0 4 1 106 10 μL or 100 μL well Adherent 5 30 100 μL 0 5 1 106 100 μL well 60 mm Suspension 5 30 10 200 100 μL 5 mL 1 2 5 ...

Page 27: ...ted without a gap see figure on the left for trouble free pipetting and proper electrical connection O ⅹ 10 Press the push button on the Neon Pipette to the first stop and immerse the Neon Tip into the cell DNA siRNA mixture Slowly release the push button on the pipette to aspirate the cell DNA siRNA mixture into the Neon Tip Note Avoid air bubbles during pipetting as air bubbles cause arcing duri...

Page 28: ...ore delivering the electric pulse Note Monitor the Neon 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 Neon Pipette fro...

Page 29: ...and Maintenance Clean the surface of the Neon device and Neon Pipette Station with a damp cloth Do not use harsh detergents or organic solvents to clean the unit The Neon Pipette is permanently calibrated at the manufacturer and does not require any further calibration Important Avoid spilling any liquid inside of the Neon Pipette Station to prevent any build up of rust on the ball plunger in the ...

Page 30: ...er prepared as described in pages 16 17 High quality DNA at a concentration of 1 5 μg μL in deionized water or TE buffer or high quality RNAi duplex at a concentration of 100 250 μM in nuclease free water page 13 Cell culture plates containing the appropriate medium Workflow General workflow for optimization is described below For detailed protocols see the next page Optimization for plasmid 1 Per...

Page 31: ...Neon database as cell culture conditions may vary between laboratories Cell Type Cell Line Primary Cells Adherent Suspension Suspension Adherent Resuspension Buffer R Resuspension Buffer T Resuspension Buffer R Resuspension Buffer R Examples include 3T3 L1 HEK293 COS 7 C2C12 HeLa HCT 15 PC 12 MDCK Raw264 7 U 2OS Examples include T cells B cells NK cells PBMC Monocytes Examples include Neuronal Ste...

Page 32: ...ate Suspension 2 105 well 1 μg DNA well 30 μg plate 100 pmol in 10 μL tip 200 nM per well 10 μL well 270 μL plate 3 Set up a Neon Tube with 3 mL Electrolytic Buffer use Buffer E for 10 μL Neon Tip and Buffer E2 for 100 μL Neon Tip into the Neon Pipette Station containing the cell DNA siRNA mixture as described on page 15 4 Press Optimization and load the optimization protocols to begin electropora...

Page 33: ...e samples 10 fold in 900 μL medium and transfer 100 μL of the sample to 0 4 mL prewarmed culture medium 6 Repeat Steps 3 5 for the remaining samples 7 Gently rock the plate to assure even distribution of the cells Incubate the plate at 37 C in a humidified CO2 incubator 8 Assay samples to determine the transfection efficiency e g fluorescence microscopy or functional assay or gene knockdown for si...

Page 34: ...ation parameters in the Input window and perform electroporation using the parameters listed below Results Sample Well no Pulse Voltage Pulse Width Pulse no Transfection Efficiency Cell Viability 1 A1 Use pre optimized parameter or control without electroporation 2 A2 2000 20 1 3 A3 2050 20 1 4 A4 2100 20 1 5 A5 2150 20 1 6 A6 2200 20 1 7 B1 2250 20 1 8 B2 2300 20 1 9 B3 2350 20 1 10 B4 2400 15 1 ...

Page 35: ...on Tip see table below For using the 100 μL Neon Tip in 24 well format adjust the amounts listed in the table below appropriately by 10 fold Cell Type Format Cell no DNA siRNA Resuspension Buffer Adherent 24 well 1 105 well 0 5 μg DNA well 15 μg plate 50 pmol in 10 μL tip 100 nM per well Buffer R 10 μL well 285 μL plate Suspension 24 well 2 105 well 1 μg DNA well 30 μg plate 100 pmol in 10 μL tip ...

Page 36: ...mmediately remove the Neon Pipette and transfer the samples from the 10 μL Neon Tip into prewarmed 0 5 mL culture medium For 100 μL Neon Tip dilute samples 10 fold in 900 μL medium and transfer 100 μL of the sample to 0 4 mL prewarmed culture medium 6 Repeat Steps 3 5 for the remaining samples 7 Gently rock the plate to assure even distribution of the cells Incubate the plate at 37 C in a humidifi...

Page 37: ... 24 well format adjust the amounts listed in the table below appropriately by 10 fold Cell Type Format Cell no DNA siRNA Resuspension Buffer Adherent 24 well 1 105 well 0 5 μg DNA well 15 μg plate 50 pmol in 10 μL tip 100 nM per well Buffer R 10 μL well 285 μL plate Suspension 24 well 2 105 well 1 μg DNA well 30 μg plate 100 pmol in 10 μL tip 200 nM per well Buffer R 10 μL well 270 μL plate Primar...

Page 38: ...g DNA but no electroporation pulse 5 After electroporation immediately remove the Neon Pipette and transfer the samples from the 10 μL Neon Tip into prewarmed 0 5 mL culture medium For 100 μL Neon Tip dilute samples 10 fold in 900 μL medium and transfer 100 μL of the sample to 0 4 mL prewarmed culture medium 6 Repeat Steps 3 5 for the remaining samples and incubate the plate 7 Assay samples to det...

Page 39: ...correctly Make sure that the Neon Tip is inserted into Neon Pipette correctly as described on page 20 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 Neon Tube The electrode in the tube must be completely immersed in buffer Be sure to add sample in Resuspension Buffer ...

Page 40: ...ality and impairs their physical integrity Poor optimization of electrical parameters Perform optimization for your cell type as described on page 22 Poor plasmid DNA quality or the plasmid DNA is low Use high quality plasmid DNA for transfection see page 13 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 1...

Page 41: ...p for air bubbles Remove the solution and aspirate the sample into the tip again without any air bubbles Press OK to exit the screen Please enter user name All protocols in the database need a user name Enter the user name and press OK to exit the screen Please enter protocol name All protocols in the database need a protocol name Enter the user name and press OK to exit the screen Password incorr...

Page 42: ...e 39 for a decontamination protocol and to obtain a Returns Goods Authorization RGA number and return shipping 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 bo...

Page 43: ...of Protection IPX0 Protective Earthing Class I earthed Installation Category II Instrument Type Benchtop unit Device Dimensions 9 2 inches w 11 8 inches l 8 66 inches h Pipette Station Dimensions 5 91 inches diameter 5 51 inches h Device Weight 13 2 pounds 6 kg Built in Features Touch screen 800 480 pixels digital display The Neon Transfection System including the Neon Pipette Station is compatibl...

Page 44: ...oltage matches the voltage available in your location For operating environment see page 35 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 mai...

Page 45: ...cates 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 Visit www invitrogen com weee for collection and recycling options The CE mark symbolizes that the product conforms to all applicable European Community provisions for which this marking is required Operation...

Page 46: ...rat 5 nmol AM4649 Silencer FAM labeled GAPDH siRNA human mouse rat 5 nmol AM4650 Countess Automated Cell Counter 1 each C10227 PureLink HiPure Plasmid Miniprep Kit 25 preps K2100 02 PureLink HiPure Plasmid Midiprep Kit 25 preps K2100 04 PureLink HiPure Plasmid Filter Midiprep Kit 25 preps K2100 14 PureLink HiPure Plasmid Maxiprep Kit 25 preps K2100 07 PureLink HiPure Plasmid Filter Maxiprep Kit 25...

Page 47: ...603 7200 Tel Toll Free 1 800 955 6288 Fax 1 760 602 6500 E mail tech_support invitrogen com Japanese Headquarters LOOP X Bldg 6F 3 9 15 Kaigan Minato ku Tokyo 108 0022 Tel 81 3 5730 6509 Fax 81 3 5730 6519 E mail jpinfo invitrogen com European Headquarters Inchinnan Business Park 3 Fountain Drive Paisley PA4 9RF UK Tel 44 0 141 814 6100 Tech Fax 44 0 141 814 6117 E mail eurotech invitrogen com SDS...

Page 48: ...oduct or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research For products that are subject to multiple limited use label licenses the terms of the most restrictive limited use label l...

Page 49: ... Invitrogen THIS WARRANTY AND THE REMEDIES SET FORTH HEREIN ARE EXCLUSIVE AND IN LIEU OF ALL OTHER EXPRESS OR IMPLIED WARRANTIES INCLUDING IMPLIED WARRANTIES OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT AND NO OTHER WARRANTIES SHALL BE BINDING UPON LIFE TECHNOLOGIES CORPORATION IN NO EVENT SHALL LIFE TECHNOLOGIES CORPORATION BE LIABLE FOR ANY SPECIAL INCIDENTAL OR CONSEQ...

Page 50: ...42 Notes ...

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Page 52: ...rporate Headquarters 5791 Van Allen Way Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech_support invitrogen com For country specific contact information visit our web site at www invitrogen com ...

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