Neon Transfection System - Thermo Fisher Scientific
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1. e 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 10 200 nM Note The siRNA concentration in the Neon transfection protocol refers to the siRNA concentration in the culture medium and not to the siRNA concentration in the electroporation mix in the Neon Tip Neon Transfection System Set up the Neon 1 Ensure the Neon Pipette Station is connected to the Neon device page 6 Pipette Station 2 Fill the Neon Tube with 3 mL of Electrolytic Buffer use Buffer E for 10 pL Neon Tip and Buffer E2 for 100 pL Neon Tip Note 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 4 The station is ready for use Proceed to preparing cells next page Neon Transfection System 15 Prepare adherent cells 16 10 11 Cultivate the required number of cells see below One two days prior to electroporation transfer the cells into flask with fresh growth medium such that the cells are 70 90 confluent on the day of the experiment 5 x 10 2 x 10 cells per2. Optimization Cleaning and maintenance 17 Repeat Steps 7 16 for the remaining samples Be sure to change the Neon Tips after using it twice and Neon Tubes after 10 usages Use a new Neon Tip and Neon Tube for each new plasmid DNA sample 18 Gently rock the plate to assure even distribution of the cells Incubate the plate at 37 C in a humidified CO incubator 19 If you are not using the Neon 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 Based on your initial results you may need to optimize the electroporation parameters for your cell type See page 22 for using the 18 well or preprogrammed 24 well optimization protocol on the Neon device 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 pipette station In case you accidentally spill any liquid e g buffer water coffee inside the Neon Pipette Station disconnect the station from the main device and wipe the station using dry laboratory paper Invert and allow the stat
3. Ramos U 937 as well as primary adherent cells such as neuronal cells stem cells hepatocytes HUVEC macrophage cells dendritic cells Resuspension Buffer T Use Resuspension Buffer T with primary blood derived suspension cells such as T cells B cells NK cells PBMC monocytes Neon Transfection System DNA quality and amount siRNA quality and amount Controls 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 PureLink HiPure Plasmid DNA Purification Kits page 40 to prepare DNA e Resuspend the purified DNA in deionized water or TE buffer 10 mM Tris HCL 1 mM EDTA pH 8 0 at a concentration between 1 5 ug uL Concentrations may vary depending on cell type e The DNA amount should not exceed 10 of total volume used e Check the purity of the purified DNA preparation by measurement of the A560 280 ratio The ratio should be at least 1 8 for electroporation e The device has been routinely tested with 4 7 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 The quality and concentra
4. 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 uL or 100 pL Mounting 7 stem gt Piston Gold electrode Tip Neon Transfection System Getting started Install the Neon Le Device with Pipette 2 Station Methods Unpack the Neon device as instructed on page vi Four power cords are shipped with the device to ensure that the cord you use is compatible with your local socket format Place the Neon device on a level laboratory bench Keep the area around the unit clear to ensure proper ventilation of the unit Note The Neon device has a small footprint and can be easily set up in the tissue culture hood for convenience For your safety Position the device properly such that the power switch and AC inlet located on the rear of the unit page vii are easily accessible Be sure to position the device such 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 Place the Neon Pipette Station near the Neon device 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 device respectively Be sure to align the ridge indic
5. 100 uL 96 tips 100 uL Neon Tubes 5 20 5 20 Resuspension Buffer R 1 mL 3x1mL 10 mL 30 mL Proprietary Resuspension Buffer T 1 mL 3x1mL 10 mL 30 mL Proprietary Electrolytic Buffer E 75 mL 2 x 150 mL Not applicable Not applicable Proprietary Electrolytic Buffer E2 Proprietary Not applicable Not applicable 75 mL 2 x 150 mL Unpacking the Neon Transfection System Unpacking instructions vi Follow the instructions below to unpack the Neon Transfection System The weight of the Neon device is 13 2 pounds 6 kg 1 Cut plastic tapes and remove the outer box Save the outer box and other packaging material in case you need to transport or ship the unit 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 Remove the Neon device and Neon pipette station from the box and place on a flat level surface Set up the Neon Transfection System as described on page 6 Neon Transfection System Front view The front view of the Neon device is shown below Touchscreen Rear view The rear view showing various parts of the Neon device are shown below 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 th
6. 15 C3 2100 15 2 16 C4 2150 15 2 17 C5 2200 15 2 18 C 2250 15 2 After electroporation immediately remove the Neon Pipette and transfer samples from the 10 pL Neon Tip into prewarmed 0 5 mL culture medium Repeat Steps 3 5 for the remaining samples Gently rock the plate to assure even distribution of the cells Incubate the plate at 37 C in a humidified CO incubator 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 next page Neon Transfection System 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 on the next page perform optimization using these narrower parameters as below 1 Make sure you have cells prepared as described on pages 16 17 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 pL Neon Tip see table below For using the 100 pL Neon Tip in 24 well format adjust the amounts listed in the table below appropriately by 10 fo
7. 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 to the Neon Tip Wrong buffers used High voltage connector is not connected Use the Electrolytic Buffer Buffer E for 10 uL tip and Buffer E2 for 100 yL tip in the Neon Tube and the sample in Resuspension Buffer in the Neon Tip Do not switch buffers or use any other buffer as these buffers are specifically designed for electroporation with the Neon device Be sure to connect the high voltage connector of the Neon Pipette Station to the high voltage port on the rear of the Neon device page 6 If the error persists and all connections are correct Perform self diagnostics test Perform self diagnostics test by clicking on Y 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 page 41 Neon Transfection System 31 Problem Cause Solution Arcing sparks Air bubbles in the Avoid any air bubbles in the Neon Tip while aspirating the Neon Tip
8. fold in 900 pL medium and transfer 100 uL of the sample to 0 4 mL prewarmed culture medium Repeat Steps 3 5 for the remaining samples Gently rock the plate to assure even distribution of the cells Incubate the plate at 37 C in a humidified CO incubator 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 next page Neon Transfection System 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 10 200 nM 1 Make sure you have cells prepared as described on pages 16 17 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 Neon Transfection System 2 For each electroporation sample using the 10 pL Neon Tip see table below For using the 100 pL 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 Resuspensicn Buffer Buffer R Adherent 24 well 1x 10 well ee ee DAS ae 10 pL well Hoek P 285 uL plate Buffer R Suspension 24 well 2 x 10
9. instrument is used in a manner not specified by the manufacturer Neon Transfection System 37 Informations de s curit S curit 38 Suivez les instructions de cette section pour vous assurer d utiliser l appareil Neon Transfection en toute s curit Le Neon Transfection System est con u pour r pondre aux normes de s curit EN61010 1 Pour assurer un fonctionnement s r et fiable utilisez toujours le Neon Transfection System conform ment aux instructions de ce manuel Le non respect des instructions contenues dans ce manuel pourrait engendrer un ventuel danger pour la s curit et annulerait la garantie du fabricant ainsi que la certification la norme de s curit NF EN61010 1 Life Technologies ne peut tre tenu responsable de toute blessure ou dommage provoqu s par l utilisation de cet instrument dans des buts autres que ceux pr vus Toutes les r parations et la maintenance doivent tre effectu es par Life Technologies e Assurez vous toujours que la tension d entr e de l alimentation corresponde la tension disponible sur le lieu d utilisation e Pour l environnement d exploitation consultez la page 35 e Cet appareil tant a ror frig r ses surfaces chauffent lorsqu il fonctionne Lors de l installation de l appareil laissez un espace sup rieur 10 cm 4 pouces autour de celui ci e N introduisez jamais d objets m talliques dans les orifices d a ration de l appareil car c
10. pM in nuclease free water page 13 e Cell culture plates containing the appropriate medium General workflow for optimization is described below For detailed protocols see the next page 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 Neon Transfection System Choose the appropriate optimization protocol Cell Line Adherent Buffer R Examples include 3T3 L1 HEK293 COS 7 C2C12 HeLa HCT 15 PC 12 MDCK Raw264 7 U 20S Neon Transfection System Suspension Resuspension Buffer R Examples include CEM HL 60 K 562 Jurkat LCL Ramos U 937 Cell Type Examples include Neuronal Stem Hepatocyte HUVEC Macrophage Dendritic Primary Cells Based on your cell type choose the appropriate optimization protocol as shown below Optimizations are generally required for cell types which are not in the Neon database
11. sample High voltage or pulse Reduce the voltage or pulse length settings length settings Accidentally used Do not precipitate DNA with ethanol to concentrate DNA as salt precipitated it can cause arcing due to salt contamination DNA Low cell Poor DNA quality Use high quality plasmid DNA for transfection see page 13 survival rate Low transfection 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 Neon Tip Poor optimization of Do not use the same Neon Tip for electroporation for more than 2 times because the repeated application of electric pulses reduce the tip quality and impairs their physical integrity Perform optimization for your cell type as described on efficiency electrical parameters page 22 Poor plasmid DNA Use high quality plasmid DNA for transfection see page 13 quality or the for guidelines and recommendations on DNA quality plasmid DNA is low Start with 0 5 ug plasmid DNA per sample Incorrect cell density Cell densities gt 3 x 10 or lt 5 x 10 per sample drastically reduc
12. specified protocols See page 3 for details e Neon Pipette Station The Neon Pipette Station is a unique component of the system and holds the Neon Pipette during electroporation and protects the user from any electrical shock exposures The Neon Tube which has an electrode near the bottom is inserted into the pipette station to transfer the electric field from the electrode inside the Neon Tip See page 3 for details e Neon Kits not supplied with the device The Neon Kits contain the Neon Tips Neon Tubes and buffers for electroporation The Neon Kits are available in two formats for electroporation of 10 pL or 100 pL samples page 40 for ordering information See page 3 for details on Neon Tips and Tubes Neon Transfection System System overview Features Unlike standard cuvette based electroporation the Neon Transfection System uses a unique electroporation reaction chamber the Neon Tip that delivers a high electric field to the biological sample The Neon 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
13. the tip again without any air bubbles Press OK to exit the screen Please enter user name Please enter protocol name 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 Password incorrect please re enter Re enter the 4 digit password and press OK to exit the screen Input voltage pulse width or pulse number error Neon Transfection System 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 33 Appendix Repackaging the instrument Repackaging and If you need to send the device to Invitrogen for warranty issues or you wish to storage transport the instrument to another location repackage the unit as follows instructions 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 page 41 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 de
14. well Le M eras Una 10 pL well HYP P 270 uL plate Primary Buffer T Suspension 18 well 1 2 x 105 well Fo A RU es 10 uL well Blood Cells H9 P P 180 uL plate 3 Set up a Neon Tube with 3 mL Electrolytic Buffer use Buffer E for 10 pL Neon Tip and Buffer E2 for 100 uL Neon Tip into the Neon Pipette Station and Neon Tip containing the cell DNA siRNA mixture 4 Perform electroporation using the parameters listed on the next page 29 Optional Optimization protocol Day Three continued 30 Sample Well no Pulse Pulse Pulse Rens voltage width no Transfection efficiency Cell viability 1 Al 1450 10 2 2 A2 1475 10 2 3 A3 1500 10 2 4 A4 1525 10 2 5 A5 1550 10 2 6 A6 1575 10 2 7 B1 1375 10 3 8 B2 1400 10 e 9 B3 1425 10 3 10 B4 1450 10 3 11 B5 1475 10 3 12 B 1500 10 3 13 C1 Control containing DNA but no electroporation pulse After electroporation immediately remove the Neon Pipette and transfer the samples from the 10 uL Neon Tip into prewarmed 0 5 mL culture medium For 100 uL Neon Tip dilute samples 10 fold in 900 pL medium and transfer 100 uL of the sample to 0 4 mL prewarmed culture medium Repeat Steps 3 5 for the remaining samples and incubate the plate Assay samples to determine the transfection efficiency e g fluorescence microscopy or functional assay or gene knockdown for siRNA Select the best conditions and sa
15. Pa OUIDE Invitrogen by Life technologies Neon Transfection System For transfecting mammalian cells including primary and stem cells with high transfection efficiency Catalog Number MPK5000 Document Part Number 25 1055 Publication Number MANO001557 Revision A 0 For Research Use Only Not for use in diagnostic procedures technologies For Research Use Only Not for use in diagnostic procedures Information in this document is subject to change without notice DISCLAIMER LIFE TECHNOLOGIES CORPORATION AND OR ITS AFFILIATE S DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE OR NON INFRINGEMENT TO THE EXTENT ALLOWED BY LAW IN NO EVENT SHALL LIFE TECHNOLOGIES AND OR ITS AFFILIATE S BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF Important Licensing Information This product may be covered by one or more Limited Use Label Licenses By use of this product you accept the terms and conditions of all applicable Limited Use Label Licenses 2014 Thermo Fisher Scientific Inc All rights reserved All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specifi
16. Voltage Width Pulse Displays the electroporation parameter for each protocol e Load Function buttons Used to load a protocol The Load button is activated only after a protocol is selected e Page scroll To scroll to next or previous page 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 CAD Optimization Well3 1500 Optimization Wella 1600 The electroporation parameters are displayed on the start up screen Neon Pipette Station for electroporation page 14 Upgrades for the Neon device firmware are available To download Neon device firmware upgrades go to www lifetechnologies com neon Follow instructions on the page to download the upgrades Neon Transfection System 11 General guidelines Recommended kits Recommended buffers 12 To use the Neon device for electroporation of mammalian cells you need to purchase the Neon Kits Ordering information is on page 40 Do not use any other kits with the unit To obtain the best results follow these recommendations e 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 e Before using the device with your samples ensure that you are able to insert and use the Neon Pip
17. age 27 Neon Transfection System 25 18 well optimization protocol for primary suspension blood cells Day One Make sure you have cells prepared as described on pages 16 17 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 pL Neon Tip in 18 wells of a 24 well plate see table below A Resuspension Cell type Cell no DNA siRNA Buffer T Primary blood 2 x 105 well 1 ug DNA well 100 pmol in 10 uL tip 10 pL well suspension cells is 20 ug plate 200 nM per well 180 pL plate 3 4 Set up a Neon Tube with 3 mL Electrolytic Buffer E into the Neon Pipette Station and Neon Tip containing the cell DNA siRNA mixture Input the electroporation parameters in the Input window and perform electroporation using the parameters listed below 26 Sample Well no Pulse Pulse Pulse Resulis ere voltage width 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 11 B5 2450 15 1 12 B 2500 15 1 13 C1 2000 15 2 14 C2 2050 15 2
18. and providing an ergonomic workflow The transfection occurs in the uniquely designed Neon 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 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 Important features of the Neon Transfection System are listed below e User friendly Neon 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 e Ability to transfect 1 x 10 5 x 10 cells per reaction in a sample volume of 10 pL or 100 pL in a variety of cell culture formats 60 mm 6 well 48 well and 24 well e Utilizes a single buffer system for all cell types except primary suspension blood cells e Simple touch screen interface for easy programming of electroporation parameters e Available with one pre programmed 24 well optimization protocol and the option to customize up to 50 cell specific protocols e Built in safety features in the device to enhance user safety Neon Transfection System Description of parts Neon Device The Neon device is a simple user friendly benchto
19. as tre mis au rebut avec des d chets m nagers non tri s Suivez la r glementation locale relative l limination des d chets usuels pour r duire l impact environnemental des DEFE Rendez vous sur www invitrogen com weee pour prendre connaissance des options de collecte et de recyclage M M La marque CE est un symbole indiquant que le produit est conforme toutes les dispositions applicables de la Communaut europ enne pour lesquelles ce marquage est obligatoire L utilisation du Neon Transfection System est soumise aux conditions d crites dans ce manuel Si vous utilisez l instrument d une mani re non sp cifi e par le fabricant la protection offerte par l appareil pourrait s en trouver d t rior e Neon Transfection System 39 Accessory products Additional products The following products are for use with the Neon Transfection System and are available separately For more information visit www lifetechnologies com or contact Technical Support page 41 Product Quantity Catalog no Neon Kit 10 pL 1 kit 50 reactions MPK1025 1 kit 192 reactions MPK1096 Neon Kit 100 pL 1 kit 50 reactions MPK10025 1 kit 192 reactions MPK10096 Neon Pipette 1 each MPP100 Neon Pipette Station 1 each MPS100 Neon Tubes 1 pack of 100 MPT 100 ma ee LS Saline D PBS 1X liquid 500 mL 14190 144 BLOCK iT Fluorescent Oligo for
20. ated by a white arrow on the sensor connector on the Neon Pipette Station with a groove indicated by a white dot on the sensor port of the Neon device see figure below for details IMPORTANT To connect or disconnect the sensor connector to the Neon device always handle the sensor connector using the cord plug and not the cord cable Ensure the AC power switch is in the Off position page vii Neon Transfection System Register the device Electroporation protocol options Input values limit 8 Attach the power cord to the AC inlet on the rear of the Neon device and then to the electrical outlet Use only properly grounded AC outlets and power cords o 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 next page 10 The Neon 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 finger tip or a touch screen pen See next page for details You are ready to use the Neon Transfection System See page 14 for details Visit www lifetechnologies com neon to register the device and activate your warranty or extended warranty and ensure that you receive product updates special offers and faster service There are three options to select an electroporation protocol for your cell type e If you already have the ele
21. but may also be needed for cell types that exist in the Neon database as cell culture conditions may vary between laboratories Suspension Resuspension Resuspension Buffer R Buffer T Examples include T cells B cells NK cells PBMC Monocytes 23 24 well 1 Make sure you have cells prepared as described on pages 16 17 have the DNA optimization or siRNA and prepare a 24 well plate containing 0 5 mL culture medium with protocol for serum and without antibiotics to transfer the electroporated cells Prepare adherent and enough cells and plasmid DNA siRNA for at least 30 transfections 2 2 For each electroporation sample using the 10 pL Neon Tip in 24 well Suspension cell format see table below For using the 100 uL Neon Tip in 24 well format lines Day One adjust the amounts listed in the table below appropriately by 10 fold Cell type Cell no DNA siRNA jar Adherent 1x 10 well 0 5 ug DNA well 50 pmol in 10 pL tip 10 pL well 15 ug plate 100 nM per well 285 uL plate Suspension 2x 10 well 1 yg DNA well 100 pmol in 10 uL tip 10 pL well 30 ug plate 200 nM per well 270 pL plate 3 Set up a Neon Tube with 3 mL Electrolytic Buffer use Buffer E for 10 pL Neon Tip and Buffer E2 for 100 uL Neon Tip into the Neon Pipette Station containing the cell DNA siRNA mixture as described on page 15 4 Press Optimization a
22. ce 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 Neon Transfection System Informational symbols The symbols used on the Neon Transfection System and in the manual are explained below A The Caution symbol denotes a risk of safety hazard Refer to the accompanying documentation ON power OFF power D O Protective earth ground Used on the instrument to indicate an area where a potential shock hazard may exist A s WEEE Waste Flectrical 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 Visit www lifetechnologies com weee for collection and recycling options CE The CE mark symbolizes that the product conforms to all applicable European Community provisions for which this marking is required Operation of the Neon Transfection System is subject to the conditions described in this manual The protection provided by the device may be impaired if the
23. ctroporation parameters for your cell type input the parameters in the Input Window see below e 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 page 9 You can also view our library of protocols for commonly used cell types from www lifetechnologies com neon and in put the parameters in the Database Window see below for various cell types e If you do not have any specific electroporation parameters for your cell type and wish to perform optimization use the Optimization Window page 11 The Neon 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 500 2 500 V Input Pulse Width range 1 100 ms Input Pulse Number range 1 10 Neon Transfection System 7 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 on the rear side of the unit page vii to turn ON the Neon device The unit checks to ensure that the Neon Pipette Station is connected to the device and then the start up screen is displayed User optimization Optimization A Neon _ ee amp er Protocol Weli Ov Li vor ims Pulses 1 pulses 2 Press Voltage to activate the number key pad to input voltage value Press
24. e high voltage and sensor connector of the Neon Pipette Station to the unit Sensor port High voltage AC inlet port Power switch USB port panel Fan vii Neon Pipette The Neon Pipette Station is supplied with a high voltage and sensor connector which connects the pipette station to the Neon device The Neon Pipette with a Station ee A a i Neon Tip 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 e The touchscreen buttons to operate the device e The Digital Display that shows the protocol that is in use and various parameters of the protocol Useiotmiton Em Q protocol Na Digital display ims Touchscreen buttons 1 pulses viii Introduction About the product Neon Transfection System System components The Neon 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 Neon 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 transfec
25. e push button on the pipette to the second stop to open the clamp Neon Transfection System Electroporation protocol continued 8 Insert the top head of the Neon Pipette into the Neon Tip until the clamp fully picks up the mounting stem of the piston see below gt Top head i 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 Neon Pipette and Tip are tightly connected without a gap see figure on the left for trouble free pipetting and proper electrical connection 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 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 air bubbles H Neon Transfection System 19 Electroporation 11 protocol continued 12 13 14 15 16 20 Insert the Neon Pipette with the sample vertically into the Neon Tube placed in the Neon Pipette Station until you hear a click sound Ensure that the pipette project
26. each 10 pL Neon Tip for most optimized protocols 5 x 10 2 x 10 cells per each 100 uL Neon Tip for most optimized protocols Pre warm an aliquot of culture medium containing serum PBS without Ca and Mg and Trypsin EDTA solution to 37 C Aspirate the media from cells and rinse the cells using PBS without Ca and Mg Trypsinize the cells using Trypsin EDTA or TrypLE Express Cat no 12563 After neutralization harvest the cells in growth medium with serum 0 75 mL for 10 uL Neon Tip or 7 5 mL for 100 uL Neon Tip Take an aliquot of trypsinized cell suspension and count cells to determine the cell density Transfer the cells to a 1 5 mL microcentrifuge tube or a 15 mL conical tube and centrifuge the cells at 100 400 x g for 5 minutes at room temperature Wash cells with PBS without Ca and Mg by centrifugation at 100 400 x g for 5 minutes at room temperature Aspirate the PBS and resuspend the cell pellet in Resuspension Buffer R at a final density of 1 0 x 107 cells mL Gently pipette the cells to obtain a single cell suspension Note Avoid storing the cell suspension for more than 15 30 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 page 18 or optimization protocols pages 24 29 Prepare 24 well plates by filling the wells with 0 5 mL o
27. ed Cy is a registered trademark of GE Healthcare UK Limited TaqMan is a registered trademark of Roche Molecular Systems Inc used under permission and license Contents Produc CONTEN S caia e rt aad Me e ts v Unpacking the Neon Transfection A dia die ln ni eal ar a tte ire vi Neon Transfection System 2 sleep un dre ne ld a ee neo tt vil Introduction Renae PRR Ser Reece e MERA See ee ne AE ene eee ene tue 1 Ab utthe producte frs oo tee es eee es ee ee eee Od Rice eee ae cee ee 1 Description of parts sis eee ee ee ee ees 3 METROS arar vise dues nan hese seine rad idee ET 6 Getting Sstarted sorom E see een eras een en nue oe ee reece 6 General guidelines 2 ipisna tte at ta ted to dent tag et 12 Using the Neon Transfection stat Met Ant ne en nn 14 Optimization protocol for DNA and siRNA ie 22 TroubleshootiNg nanea min ane ica 31 Neon DEVICE Promesa deg Len en eck ta tht aes th actA eue 33 Appendiks raaa CRUE PAR ste RE Qt a at a a Salata aa 34 Repackaging the instrument ss 34 Product speaitications issue eh AN ted ted te rel ca bal ha fail Ned Cab EAE ren 35 Safety iNfOrMmatlOn tel sae cd elas tele cel teak nu Poele in dd o pa nie pa o ls os 36 Informations d S curit antennes Giese Gee at a a ls ul 38 NAT siennes 40 Technical Supporte ts eh oe lll de el le lada dl cis lado dal Pete 41 iii iv Product contents Upon receiving the device Neon Transfection System Examine the unit ca
28. ela pourrait provoquer un choc lectrique des blessures corporelles ou endommager l quipement e Mettez toujours le commutateur principal de l alimentation sur OFF ARRET avant de brancher le cordon d alimentation sur la prise murale e V rifiez toujours que la borne de mise la terre de l appareil et celle de la prise murale sont correctement raccord es Branchez le cordon d alimentation sur une prise d alimentation a 3 conducteurs et reli e a la terre e Pour viter tout risque potentiel de choc lectrique v rifiez que le cordon d alimentation est correctement reli a la terre e Veillez placer l instrument de mani re pouvoir le d brancher facilement e Veillez mettre le commutateur principal sur OFF ARR T d brancher le cordon d alimentation et immobiliser la station pipettes avant de d placer l appareil Neon Transfection System Symboles d information Les symboles utilis s sur le Neon Transfection System et dans le manuel sont expliqu s ci dessous A Le symbole Attention indique un risque d accident Consultez la documentation fournie ON MARCHE alimentation OFF ARR T alimentation O Protection par la mise la terre masse Utilis e sur l instrument pour indiquer une zone o existe un risque potentiel d lectrocution A Le symbole DEEE D chets d quipements lectriques et lectroniques indique que ce produit ne doit p
29. electroporation 75 uL 13750062 ere GAPDH Positive Control siRNA human Aniol 4390849 Silencer Select negative Control No 1 siRNA 40 nmol 4390844 Silencer Cy 3 labeled GAPDH siRNA human mouse 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 preps K2100 17 MagMax 96 Total RNA Isolation Kit 96 reactions AM1830 TaqMan Gene Expression Cells to CT Kit 100 reactions AM1728 alamar Blue 25 mL DAL1025 Cell culture media A large variety of cell culture media and products for mammalian cells including primary and stem cells is available from Invitrogen For more information visit www lifetechnologies com or contact Technical Support page 41 siRNA A large variety of siRNA products including Stealth RNAi Silencer Select RNAi Silencer RNAi or standard unmodified siRNA is available from Invitrogen For more information visit www lifetechnologies com rnai or contact Technical Support page 41 40 Neon Transfection System Technical support Obtaining support For the latest services and support information for all locations go to www lifetechno
30. es transfection efficiency Use 5 x 101 5 x 10 cells per 10 pL per sample Mycoplasma Test cells for Mycoplasma contamination contaminated cells Start a new culture from a fresh stock Non Inconsistent cell Always use cells with low passage number and harvest cells reproducible confluency or with comparable confluency levels transfection passage number efficiency Multiple use of Do not use the same Neon Tip for more than 2 times Neon Tip and because the repeated application of electric pulses reduce the Neon Tube tip quality and impairs their physical integrity Do not use the same Neon Tube for more than 10 times Always use a new Neon Tip and Neon Tube for different plasmid DNA samples to avoid any cross contamination High energy Used high electrical Set lower voltage or duration error parameters 32 Neon Transfection System Neon Device error messages Introduction 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 page 41 if you need to send the device for servicing Error message Action Please connect station The Neon Pipette Station is not connected properly ensure that the sensor connector is connected to the sensor port on the rear of the device page 6 Check tip for air bubbles Remove the solution and aspirate the sample into
31. ette and Tip correctly into the Neon Pipette Station see page 14 for details e Wear gloves laboratory coat and safety glasses during electroporation e Always use the Neon device with Neon Kits for electroporation of mammalian cells e The Neon Transfection System is compatible for use with most mammalian cells including primary and stem cells e Use high quality DNA and siRNA to obtain good transfection efficiency e Follow the guidelines on pages 16 17 for cell preparation e Use an appropriate GFP green fluorescent protein construct or siRNA control see next page for details to determine transfection efficiency e 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 e Visit www lifetechnologies com neon for a library of electroporation protocols for commonly used cell types The Neon Kits contain two Resuspension Buffers Use the appropriate Resuspension Buffer based on the cell type as below The cell specific Neon transfection protocols available on www lifetechnologies com neon indicate the type of Resuspension buffer for use with each cell type Resuspension Buffer R Use Resuspension Buffer R with established adherent and suspension cell lines such as 3T3 L1 HEK293 Cos7 C2C12 HeLa HCT 15 PC 12 MDCK Raw264 7 U 205 CEM HL 60 K 562 Jurkat LCL
32. f culture medium containing serum and supplements without antibiotics and pre incubate plates in a humidified 37 C 5 CO incubator If you are using other plate format see page 18 for plating medium volume recommendations Neon Transfection System Prepare suspension cells Cultivate the required number of cells see below One to two days prior to electroporation transfer the cells into flask with fresh growth medium such that the cells are 70 90 confluent on the day of the experiment For most cell lines the cell density is 1 3x 10 cells T 25 flask 1 5 x 10 cells per each 10 uL Neon Tip for most optimized protocols 1 5 x 10 cells per each 100 uL Neon Tip for most optimized protocols Pre warm an aliquot 500 uL per sample for 10 uL Neon Tips or 5 mL for 100 uL Neon Tips of culture medium containing serum Also prepare an appropriate volume of PBS without Ca and Mg Take an aliquot of cell culture and count the cells to determine the cell density Transfer the cells to a microcentrifuge tube or 15 mL conical tube and pellet the cells by centrifugation at 100 400 x g for 5 minutes at room temperature Wash the cells with PBS without Ca and Mg and pellet the cells by centrifugation at 100 400 x g for 5 minutes at room temperature Aspirate the PBS and resuspend the cell pellet in Resuspension Buffer R or Resuspension Buffer T at a final density of 2 0 x 10 cells mL Gently pipette the ce
33. ion is inserted into the groove of the pipette station Note Ensure the metal head of the Neon Pipette is tightly connected to the ball plunger inside of the Neon Pipette Station and to the Neon Tube see figure on the left for the correct position W Ensure that you have selected the appropriate electroporation protocol and press Start on the touchscreen The Neon device automatically checks for the proper insertion of the Neon Tube and Neon Pipette before 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 After delivering the electric pulse Complete is displayed on the touchscreen to indicate that electroporation is complete Slowly remove the Neon Pipette from the Neon Pipette Station and immediately transfer the samples from the Neon 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 To discard the Neon Tip press push button to the second stop into an appropriate biological hazardous waste container Neon Transfection System Electroporation protocol continued
34. ion to completely dry for 24 hours at room temperature Do not use the oven to dry the Neon Pipette Station If the station does not work after drying contact Technical Support page 41 For any other repairs and service contact Technical Support page 41 Do not perform any repairs or service on the Neon device yourself as it is a high voltage hazard and to avoid any damage to the unit or voiding your warranty Neon Transfection System 21 Optimization protocol for DNA and siRNA Introduction Materials needed Workflow 22 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 Neon device is preprogrammed with a 24 well optimization protocol using the 10 uL or 100 pL Neon 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 26 Ordering information is on page 40 e Neon 10 pL or 100 uL Kit e Cells in Resuspension Buffer prepared as described in pages 16 17 e High quality DNA at a concentration of 1 5 ug uL in deionized water or TE buffer or high quality RNAi duplex at a concentration of 100 250
35. ithout saving the parameters press X The database window is displayed Press the desired protocol and then press Load to load electroporation parameters from the database Proceed to preparing cells pages 16 17 and DNA and setting up the Neon Pipette Station for electroporation page 14 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 Neon Transfection System Optimization window Upgrade the firmware Perform optimization of electroporation parameters using the preprogrammed 24 well optimization protocol These protocols are locked and cannot be edited l 4 5 Proceed to preparing cells pages 16 17 and DNA and setting up the Press the power switch located on the rear side of the unit page vii to turn ON the Neon device The unit checks to ensure that the Neon Pipette Station is connected to the device and then the start up screen is displayed User optimization Gas opt Protocol C 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 e User and Protocol Displays the optimization and well number e Parameters
36. ld Celltype Format Cell no DNA siRNA Resuspensign Buffer 0 5 pg DNA well 50 pmolin 10 pL ti eee Adherent 24 well 1x 10 well l AS PA o ae 10 pL well HYP P 285 pL plate 1 ug DNA well 100 pmol in 10 pL ti PAPET Suspension 24 well 2 x 10 well 19 AN BAREA o 10 uL well 30 ug plate 200 nM per well 270 uL plate Primary l Buffer T Suspension 18 well 1 2 x 105 well de PE has DE RU Es 10 uL well Blood Cells H9 P P 180 uL plate 3 Set up a Neon Tube with 3 mL Electrolytic Buffer use Buffer E for 10 pL Neon Tip and Buffer E2 for 100 uL Neon Tip into the Neon Pipette Station and Neon Tip containing the cell DNA siRNA mixture 4 Perform electroporation using the parameters listed on the next page Neon Transfection System 27 Optimization protocol Day Two continued 28 Sample Wellno Pulse Pulse Pulse Results voltage width no Transfection efficiency Cell viability 1 Al 1450 20 1 2 A2 1475 20 1 3 A3 1500 20 ah 4 A4 1525 20 1 5 A5 1550 20 1 6 A5 1575 20 1 7 B1 1375 30 1 8 B2 1400 30 1 9 B3 1425 30 1 10 B4 1450 30 1 11 B5 1475 30 1 12 B 1500 30 1 13 C1 Control containing DNA but no electroporation pulse After electroporation immediately remove the Neon Pipette and transfer the samples from the 10 pL Neon Tip into prewarmed 0 5 mL culture medium For 100 uL Neon Tip dilute samples 10
37. lls to obtain a single cell suspension Note Avoid storing the cell suspension for more than 15 30 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 page 18 or optimization protocols pages 24 29 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 C 5 CO incubator If you are using other plate format see page 18 for plating medium volume recommendations Neon Transfection System 17 Electroporation 1 Make sure you have appropriate number of cells prepared as described on protocol pages 16 17 have the plasmid DNA or siRNA at the suggested concentrations page 13 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 page 22 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 siRNA T Vol plating Buffer R or Format Cell Type DNA ug nM Neon Tip Ne Cell no Buffer T Adherent 0 25 0 5 10 uL 1 2 x 10 10 pL
38. logies com At the website you can eAccess worldwide telephone and fax numbers to contact Technical Support and Sales facilities eSearch through frequently asked questions FAQs eSubmit a question directly to Technical Support techsupport lifetech com eSearch for user documents SDSs vector maps and sequences application notes formulations handbooks certificates of analysis citations and other product support documents eObtain information about customer training eDownload software updates and patches Safety DataSheets Safety Data Sheets SDSs are available at www lifetechnologies com sds SDS Certificate of The Certificate of Analysis provides detailed quality control and product qualification information for each product Certificates of Analysis are available on our website Go to www lifetechnologies com support and search for the Certificate of Analysis by product lot number which is printed on the box Analysis Limited Product Life Technologies and or its affiliate s warrant their products as set forth in the Life Technologies General Terms and Conditions of Sale found on the Life Technologies web site at www lifetechnologies com termsandconditions If you have any questions please contact Life Technologies at www lifetechnologies com support Warranty Neon Transfection System 41 Notes 42 Neon Transfection System For support visit lifetechnologies com support or email techsupp
39. n cells General instructions to prepare cells for use with the Neon Transfection System are described below For primary and stem cell types use the established methods developed in the laboratory See page 22 if you wish to use the preprogrammed optimization protocol Ordering information is on page 40 e Cells e Neon Kits e High quality DNA at a concentration of 1 5 ug pL in deionized water or TE buffer or high quality RNAi duplex at a concentration of 100 250 pM in nuclease free water page 13 e Cell culture plates containing the appropriate medium e D PBS or Phosphate buffered saline PBS without Ca and Mg page 40 e Trypsin EDTA or TrypLE Express Cat no 12563 for adherent cells e Countess Automated Cell Counter page 40 or equivalent 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 e To obtain the highest transfection efficiency and low non specific effects optimize transfection conditions by varying electrical parameters as described on page 22 using the pre programmed optimization protocol in a 24 well format e Since the cell culture conditions vary from user to user be sure to use low passage number actively dividing cells for dividing cells
40. nd load the optimization protocols to begin electroporation using the parameters listed below Pulse Pulse Pulse Results Sample Well no A a RTE voltage width no Transfection efficiency Cell viability 1 Al Use pre optimized parameter or control without electroporation 2 A2 1400 20 1 3 A3 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 B 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 C 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 24 Neon Transfection System 24 well optimization protocol for adherent and suspension cell lines Day One continued After electroporation immediately remove the Neon Pipette and transfer samples from the 10 uL Neon Tip into prewarmed 0 5 mL culture medium For 100 pL Neon Tip dilute samples 10 fold in 900 uL medium and transfer 100 uL of the sample to 0 4 mL prewarmed culture medium Repeat Steps 3 5 for the remaining samples Gently rock the plate to assure even distribution of the cells Incubate the plate at 37 C in a humidified CO incubator 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 p
41. on Tip Area to insert Neon Tube Neon Pipette q Connector Station 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 Neon Tip The Neon Tubes are supplied with Neon Kits as well as available separately page 40 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 A Buffer Electrode Neon Transfection System Neon Tips The Neon Tips are disposable tips composed of a tip and piston used with the Neon Pipette The Neon Tips contain a gold plated electrode to create a disposable electric chamber for the delivery of a high electric field to biological samples The Neon Tips are supplied with Neon Kits in two formats to support operating volumes of 10 pL and 100 pL respectively page 40 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
42. ortfdlifetech com lifetechnologies com 11 July 2014 technologies
43. p electroporation device When used with a Neon Pipette Station and Neon Kits the Neon device efficiently transfects mammalian cells including primary and stem 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 vii for a front and rear view of the device Neon Pipette The Neon Pipette utilizes a positive displacement pipette mechanism for pipetting mixtures A DS containing cells and nucleic acid or siRNA The AX Neon Pipette is a fixed volume pipette and 7 permanently calibrated at the manufacturing stage CH and does not require any further calibration gt The Neon Pipette is designed for use with Neon Tips only Do not use any other tips with the Neon Pipette Neon Transfection System Neon Pipette Station Neon Tube The Neon Pipette Station is a unique component of the Neon Transfection system It holds a Neon Pipette during electroporation procedures The Neon Pipette Station is equipped with many safety sensors and protection mechanisms that protect the user from any exposures to an electrical shock The Neon Pipette Station is connected to the Neon device using the high voltage and sensor connector see page 6 for details The Neon Pipette Station also holds the Neon Tube which has an electrode near the bottom that transfers the electric field from the electrode inside the Ne
44. refully 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 www lifetechnologies com neon The contents of the Neon Transfection Systems are listed below The Neon Transfection System is shipped at room temperature See page 3 for specifications and description of the Neon Transfection System and page 6 to set up the device Product Neon Transfection Device Quantity 1 Specific Power Cord for US Canada Taiwan Japan Europe and UK Neon Pipette Neon Pipette Station 4 Instruction Manual Neon Kits The Neon Kits are used with the Neon Transfection Systems for efficient transfection of mammalian cells and are available separately from page 40 The kits are available in two formats for electroporation of 10 uL and 100 uL 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 4 C and all remaining kit components at room temperature Neon Kit 10 pL Neon Kit 100 pL Item Cat no MPK1025 Cat no MPK1096 Cat no MPK10025 Cat no MPK10096 50 reactions 192 reactions 50 reactions 192 reactions Neon Tips 25 tips 10 uL 96 tips 10 uL 25 tips
45. tach 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 Invitrogen 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 C to 40 C 34 Neon Transfection System Product specifications Neon Transfection System 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 100 240 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 C to 40 C Up to 80 IPX0 Class I earthed II Benchtop unit 9 2 inches w x 11 8 inches 1 x 8 66 inches h 5 91 inches diameter 5 51 inches h 13 2 pounds 6 kg Touch screen 800 x 480 pixels digital display The Neon Transfection System including the Neon Pipette Station is compatible with standard nonhazardous laborator
46. 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 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 pages 16 17 and DNA and setting up the Neon Pipette Station for electroporation page 14 8 Neon Transfection System 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 on the rear side of the unit page vii to turn ON the Neon device The unit checks to ensure that the Neon Pipette Station is connected to the device and then
47. the start up screen is displayed N e on User optimization optimization 0 dia wie Protocol Welt 1ms Start 1 pulses 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 CT EME CHI ET ven ims Start 4 1 pulses The Database window shows Number button Indicates protocol number e User and Protocol Displays the user and protocol name e Parameters Voltage Width Pulse Displays the electroporation parameter for each protocol e 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 e Page scroll To scroll to next or previous page 3 Press the desired protocol number button to edit the protocol The selected protocol is highlighted Neon Transfection System Database window continued 10 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 a Q 2 Press Enter to save the information in the database To exit the edit screen w
48. tion is performed using as few as 1 x 10 or as many as 5 x 10 cells per reaction using a sample volume of 10 uL or 100 pL in a variety of cell culture formats 60 mm 6 well 48 well and 24 well The Neon Transfection System uses a single transfection kit Neon 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 Neon Transfection System offers open and transparent protocols that are optimized for ease of use and simplicity The Neon 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 Neon device database Optimized protocols for many commonly used cell types are also available on www lifetechnologies com neon for your convenience to maximize transfection efficiencies for your cell types See page 3 for details on various parts of the system The Neon Transfection System consists of e Neon Device The Neon Device is a simple user friendly benchtop electroporation device that 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
49. tion 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 e The recommended starting siRNA concentration is 100 250 uM in nuclease free water e The siRNA amount should not exceed 10 of total volume used 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 Clontech or equivalent For best results the vector encoding the GFP should have the following features e Strong promoter active in a variety of mammalian cells such as the immediate early CMV cytomegalovirus promoter e SV40 polyadenylation signals downstream of the GFP gene for proper processing of the 3 end of the GFP mRNA e Antibiotic selection marker e pUC origin of replication for propagation in E coli siRNA control For siRNA experiments use BLOCK iT Fluorescent Oligo for electroporation or Silencer Select GAPDH Positive Control siRNA page 40 to assess transfection efficiency Neon Transfection System 13 Using the Neon Transfection System Introduction Materials needed w Se 8 7 A AI IMPORTANT 14 Instructions are provided in this section to use the Neon device with the Neon Pipette Station and Neon Kits for electroporation of mammalia
50. ve these parameters into the database for your cell type Neon Transfection System Troubleshooting Problem Cause Solution No power the AC power cord is not Check AC power cord connections at both ends Use the correct display remains connected cords blank when the power is turned on Connection error message displayed Pipette or tube is incorrectly inserted e Properly insert the Neon Pipette into the Neon Pipette Station as described on page 20 The metal head of the pipette should be tightly connected to the ball plunger inside the pipette station e Properly insert the Neon Tube into the Neon Pipette Station as described on page 15 The side electrode on the tube should be tightly connected to the ball plunger inside the pipette station e Avoid spilling any liquid into the pipette station to prevent any build up of rust on the ball plunger in the pipette station The sensor connector is not connected e Be sure to connect the sensor connector of the Neon Pipette Station to the sensor port on the rear of the Neon device e Make sure the mark on the cable plug and the instrument connector is aligned correctly page 6 Error messages Connection failure No Neon Tip is inserted or the Neon Tip is inserted incorrectly See page 33 for a description of error messages Make sure that the Neon Tip is inserted into Neon Pipette correctly as described on page 20
51. well 96 well 10 200 100 uL Suspension 0 5 1 10 LO 2 5 x 10 10 pL well Adherent 0 25 1 10 uL 2 5 5 x 10 10 pL well 48 well 10 200 250 uL Suspension 0 5 2 10 LC 5 12 5 x 104 10 pL well Adherent 0 5 2 10 LO 0 5 1 x 10 10 pL well 24 well 10 200 500 uL Suspension 0 5 3 10 uL 1 2 5 x 10 10 pL well Adherent 0 5 3 10 uL 1 2 x 10 10 pL well 12 well 10 200 1 mL Suspension 0 5 3 10 uL 2 5 x 10 10 pL well 0 5 3 10 uL 10 uL or 100 uL 6 well 05 3 10 200 2mL 10 uL 7 10 pL or Suspension 5 30 10 pL 100 pL 0 4 1 x 10 100 pL well 100 uL Adherent 5 30 100 pL 0 5 1 x 10 100 pL well 60 mm 10 200 so mL Suspension 5 30 00 uL 1 2 5 x 10 100 pL well Adherent 5 30 mgo uL 1 2 x 10 100 uL well 10 cm 10 200 10 mL Suspension 5 30 00 uL 2 5 x 106 100 pL well Use Resuspension Buffer T for primary suspension blood cells 18 3 Set up a Neon Tube with 3 mL Electrolytic Buffer use Buffer E for 10 uL Neon Tip and Buffer F2 for 100 uL Neon Tip into the Neon Pipette Station gt page 15 Set the desired pulse conditions on the device based on your cell type page 7 Transfer the appropriate amount of plasmid DNA siRNA into a sterile 1 5 mL microcentrifuge tube Add cells to the tube containing plasmid DNA siRNA and gently mix See the above table for cell concentration DNA and plating volumes to use To insert a Neon Tip into the Neon Pipette press th
52. y reagents Do not use organic solvents in the tip tubes or with the device Neon Transfection System 35 Safety information Safety 36 Follow the instructions in this section to ensure safe operation of the Neon Transfection device The Neon Transfection System is designed to meet EN61010 1 Safety Standards To ensure safe reliable operation always operate the Neon 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 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 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 devi
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