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Gemini Twin Waveform Electroportation Systems Users`s Manual

1. Pulse Droop Error Pulse voltage droop exceeded estimated droop A Increase the sample resistance This can be achived by Increasing the gap size 2 Reducing the volume 3 Use a less conductive buffer B Reduce the pulse duration Pulse Voltage Overshoot Pulse voltage monitor p vmon pulse overshoot Hardware error Please contact the manufacturer Pulse Voltage Mon too Low Pulse voltage monitor p vmon below min voltage Hardware error Please contact the manufacturer Pulse Voltage Mon too High Pulse voltage monitor p vmon above max voltage Hardware error Please contact the manufacturer Pulse Current Mon too Low Pulse current monitor Cam p imon below min current Hardware error Please contact the manufacturer Pulse Current Mon too High Pulse current monitor p imon above max current Hardware error Please contact the manufacturer Arc_Detected Arc detected during pulsing Arc could happen due to the one or more of the following conditions Sample resistance is very low for the voltage selected 2 Gap size is too small for the voltage selected 3 Pulse duration is too long No_HB_from_GUI Communication error Hardware error Please contact the manufacturer Sample Resistance Out Of Range The measured sample load is out of range Increase the sample resistance This can be achived by Increasing the gap
2. Review parameters Position electrodes on area being electroporated To run tap the Omega icon RUN PROTOCOL HARVARD APPARATUS Rat embryo 563 EE Square Wave Set Meas 04 03 13 10 43 03 AM Voltage v 35 Duration ms 50 Resistance ohm Droop Plate of Pulses 5 Handler Pulse Interval s 1 0 Gap width mm 4 Meas Load ohm M PL HARVARD APPARATUS BTX With load measurement OK press the Go icon to run protocol RUN PROTOCOL HARYARD APPARATUS Rat embryo 563 Square Wave Set Meas 04 03 13 10 43 45 AM Voltage v 35 Resistance ohm Droop ec Plate of Pulses 5 Handler Pulse Interval s 1 0 Gap width mm 4 Meas Load ohm OK y PI lt P gt t While the pulse is being delivered the Square icon can be used to stop the protocol at any time By NOTE Generator will display readings after each pulse RUN PROTOCOL HARVARD APPARATUS Rat embryo 563 Square Wave Set Meas 02 27 13 10 36 43 AM Voltage v 35 7 Ps Gap width mm 0 Q Meas Load ohm OK LU 60 0 Resistance ohm Once protocol is complete data regarding your pulse is displayed Droop 0 3 of Pulses 5 and stored in the systems logs for future use Pulse Interval s 1 0 You may continue pulsing go back to the settings screen or the protocol select screen or to the home screen RUN PROTOCOL HARVARD APPARATUS Rat embryo 563 Square Wave S
3. amp Frey J 1998 Mechanism of electroporative dye uptake by mouse B cells Biophys J 74 98 108 Sukharev S l Klenchin V A Serov S M Chernomordik L V amp Chizmadzhev Y 1992 Electroporation and electrophoretic DNA transfer into cells The effect of DNA interaction with electropores Biophys J 63 1320 1327 Wolf H Rols M P Boldt E Neumann E amp Teissie J 1994 Control by pulse parameters of electric field mediated gene transfer in mammalian cells Biophys J 66 524 531 Zerbib D Amalric F amp Teissie J 1985 Electric field mediated transformation isolation and characterization of a TK subclone Biochem Biophys Res Commun 129 611 618 Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com 4 HARVARD APPARATUS BTX Electroporation Buffers There are many kinds of buffers and medium used for electroporating cells Typically we recommend using medias without serum or antibiotics The following is a list of the most commonly used buffers medium BTXpress is a single buffer solution developed to quickly and efficiently deliver genes into mammalian cells that were previously considered hard to transfect by chemical and other non viral methods This solution in combination with BTX electroporators provides researchers with the versatility needed for success across a broad range of ce
4. you the researchers with the tools to achieve your goals We are vested in your success To that end the Harvard Apparatus BTX technical support team constantly tracks published literature for any reference to electroporation and electrofusion We extract the pertinent experimental conditions and yields from these papers to help us in our efforts to help you In addition to tracking publications we are available to you for support at any time for advice in experimental design product recommendations troubleshooting and any other relevant technical advice We thank you again for your investment and we look forward to assisting you in any way we can Finally please read this manual carefully before attempting to operate the electroporation system If you have any questions about the unit or about particular applications please contact us Harvard Apparatus BTX 84 October Hill Rd Holliston MA 01746 USA Toll Free 1 800 272 2775 International Callers 508 893 8999 Fax 508 429 5732 Web www btxonline com Email techsupport btx harvardapparatus com For any customers outside the US or Canada please call your local Harvard Apparatus BTX dealer or call us directly HARVARD APPARATUS Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com 7 HARVARD APPARATUS BTX Electroporation Overview DNA Delivery Into Cells Using Electroporation General El
5. 1 Electrode Gap Size 2 Cell Diameter 3 Temperature General Optimization Guide for Electroporation HARVARD APPARATUS BTX Cell Types Field Strength Ranges Bacteria Yeast 3 24 kV cm Mammalian 0 25 3 kV cm Plant 3 12 kV cm Electrode Gap Size The distance between electrodes or gap size is important when optimizing your electroporation experiment Field strength is calculated using voltage divided by gap size For example using a 4mm gap cuvette with 500V would provide a field strength of 1 25kV cm If instead of a 4mm gap cuvette a 2mm gap cuvette was used the voltage would have to be reduced by half or to 250V in order to maintain the same field strength of 1 25kV cm It is possible to derive the voltage needed to accomplish electroporation if the desired field strength and gap size are known The calculation for this is Field strength kV multiplied by gap size cm equals voltage For example if a user was certain that a 1 25 kV cm field strength was required in a 1mm gap cuvette the calculation would be 1 25kV x 0 1cm 0 125kV or 125V Example A field strength of 1 25 kV cm 4mm gap cuvette 500V 2mm gap cuvette 250V 1mm gap cuvette 125V Cell Diameter Generally smaller cell sizes require higher voltages while larger cell diameters require lower voltages for successful cell membrane permeation Temperature The temperature at which cells are maintained during electroporation effects the effic
6. 4 2 2001 EN 61000 4 3 2002 EN 61000 4 4 2004 EN 61000 4 5 2001 EN 61000 4 6 2003 EN 61000 4 8 1993 A1 2000 EN 61000 4 1 1 2004 EN 61000 3 2 2000 EN 61000 3 3 2002 CISPR11 2003 A1 2013 Class A EMC and Safety compliance were evaluated by Intertek ETL Semko Reference test report file numbers 101111687 BOX 001 002 003 004 005 I the undersigned hereby declare that the equipment specified above conforms to the above Directive s and Standard s Place United States of America Date April 23 2013 C q Signature HARVARD oi APPARATUS VP Engineering Operations Position
7. At the upper limit threshold pore diameter and total pore area become too large for the cell to repair by any spontaneus or biological process The result is irreversible damage to the cell or cell lysis Another important point to consider is the generation of heat during electroporation Heat production is directly related to current intensity which is in turn dependent on the conductivity of the material through which the electric field is applied Standard saline solutions such as PBS and many tissue culture media are highly conductive and thus will generate considerable amounts of heat when used in cell electroporation Excessive heating can be detrimental to cell viability The effects of heating can be reduced by using a low conductivity medium such as BTX s Cytoporation medium to resuspend cells prior to electroporation Although electroporation is an effective method for introducing macromolecules onto cells the biological mechanisms by which cells become electroporated are not completely understood Therefore the development of specific protocols for particular applicatons is usually achieved by empirical adjustment of pulse parameters i e amplitude duration pulse number and interpulse interval Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com Glossary of Terms Capacitance The ability of a body to store an electrical charge Any object that c
8. Class Pollution Degree Installation Category ll Supplier Name Harvard Apparatus BTX Supplier Address 84 October Hill Rd Holliston MA 01746 Supplier Phone Number 508 893 8999 Regulatory Certifications CE ETL UL CSA WEEE EU RoHS amp CB Scheme Safety Declarations ANSI UL 61010 1 2012 Ed 3 Rev 2012 CAN CSA C22 2 No 61010 1 2012 Ed 3 IEC 61010 1 2001 Ed 2 Corrigendum 2002 Corrigendum 2 2003 CENELEC EN 61010 1 2001 CB Scheme EMC Declarations IEC 61326 1 Ed FCC 47 CFR Part 15 Operating Humidity vs Temperature E L E 3 x v gt E C DC 25 30 Ambient Temperature C Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com 6 A Message from Harvard Apparatus BIX Thank you for investing in a Harvard Apparatus BTX Gemini System Since its founding in 1983 BTX s main focus has been in the area of applying controlled electic fields for genetic engineering applications Because of this we quickly established a reputation as the technological leader in the fields of electroporation and electrofusion Our systems have been installed in many prestigious institutes around the globe where they are used successfully for high efficiency transfection transformation and cell fusion applications We offer a variety of waves forms electrodes and chamber options to provide
9. OK Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com 19 HARVARD APPARATUS Performing Experiments Preset Protocols continued Customizing a Preset Protocol Review parameters On the home screen tap the Preset Protocols icon Tap Omega icon to measure pre pulse load resistance HARVARD APPARATUS RUN PROTOCOL HARVARD APPARATUS 02 26 13 9 51 26 AM HEK 293 867 Square Wave Set Meas 04 02 13 4 16 48 PM Voltage v 100 Duration ms 10 Resistance ohm Droop Plate of Pulses 1 Handler Pulse Interval s Gap width mm 4 Meas Load ohm Preset Protocols User Protocols Double tap desired cell type HARVARD APPARATUS RUN PROTOCOL HARVARD APPARATUS APPARATUS 02 26 13 1 22 11 PM HEK 293 867 Square Wave Meas 04 04 02 13 4 17 14 PM 02 13 4 17 14 PM Voltage v Duration ms Resistance ohm Droop 0 3 Plate of Pulses 1 Handler Pulse Interval s Gap width mm 4 Meas Load ohm OK Scroll through available cells Once protocol is complete data regarding your pulse is displayed Tap desired cells and stored in the systems logs BTX Gemini X only for future use Tap Folder icon You may continue pulsing go back to the protocol select screen or to the home screen HARVARD APPARATUS 02 26 13 1 24 37 PM RUN PROTOCOL HARVARD APPARATUS HEK 293 867 Ee Square Wave Set Meas 04 02 13 4 17 4
10. Serial Bus Generic USB Hub hern Manufacturer Mudel Redang Des Optple 990 n Windom tapenvence indes debi Coral TM 7900 CPU Z 3 give 2 90 GH installed memory RAM 800 68 b4 bit Operstrig Sien No Pen ce Touch Input is available for this Display How do you want to search for driver software Search automatically for updated driver software Windows will search your computer and the Internet for the latest driver software for your device unless you ve disabled this feature in your device installation Browse my computer for driver software H i Locate and install driver software manually bag Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com HARVARD APPARATUS BTX Upgrading BTX Gemini Series Software USB Virtual Commport Driver Installation 6 Click Close after the software has been successfully installed continued 4 Click Browse and navigate to the generator drivers folder O A Update Drive Software USB serial port for 11 Elte Syringe Pump COMA located on the CD supplied with the generator Select the file BTX USB CDC inf Make sure the Include Subfolders box Windows has successfully updated your driver software is checked and then click Next Windows has finished installing the driver software for this device USB serial port for device e L Update Driver Software Device Browse f
11. and Biophysical Research Communications 287 pp 445 454 2001 In Vivo Electroporation Johannson D et al Intradermal Electroporation of Naked Replicon RNA Elicits Strong Immune Responses PLoS ONE 7 1 29732 2012 Daftarian P et al In vivo Electroporation and Non protein Based Screening Assays to Identify Antibodies Against Native Protein Conformations Hybridoma 30 5 2011 Hallengard D et al Comparison of plasmid vaccine immunization schedules using intradermal in vivo electroporation Clinical Vaccine Immunology 2011 Bolhassani A et al Improvement of different vaccine delivery systems for cancer therapy Molecular Cancer 10 3 2011 Li W et al The Effects of Irreversible Electroporation IRE on Nerves PLoS ONE 6 4 2011 Lladser A et al Intradermal DNA electroporation induces survivin specific CTLs suppresses angiogenesis and confers protection against mouse melanoma Cancer Immunol Immunother 59 81 92 2010 Shi W et al Generation of 5031171 transgenic RNAI mice via permanent integration of small hairpin RNAs in repopulating spermatogonial cells in vivo Acta Biochim Biophy Sci 42 2 p 116 2010 Haller BK et al Therapeutic efficacy of a DNA vaccine targeting the endothelial tip cell antigen delta like ligand 4 in mammary carcinoma Oncogene 29 4276 4286 2010 Guo Y et al Irreversible Electroporation Therapy in the Liver Longitudinal Efficacy Studies in a Rat Model
12. set the password Tap the Green Check icon to save Set The Pump Password Pumppasword Ju HARVARD APPARATUS 02 26 13 5 11 17 PM The protocol is now locked against modifications To Unlock press the lock icon Type in the password then Tap the Green Check icon HEK 293 pa OM GAP HARVARD APPARATUS 02 26 13 5 11 28 PM 11 28 PM e 3 0 Gm or Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com Touchscreen Button Reference LC N O D L L 2 EY Accept Used to accept the parameters settings on a screen and advance to the next screen in the menu also used in place of a double tap on various icons Back Used to go back one screen Cancel Used to cancel any changes entries on a screen and return to the previous screen in the menu Exponential Decay Wave Indicator Used to indicate when a protocol utilizes exponential decay wave pulses File Options Access the File Option menu that is used to save rename and delete protocols Home Main Menu Access Preset Protocols User Protocols and Settings Page Down Used to page down in a display list Page Up Used to page up in a display list Pre Pulse Resistance Measurement Used to measure the resistance of the sample prior to delivering the DC pulse Run Protocol Used to deliver the pulse protocol to the sample Scroll Down Used to scroll down in a displ
13. the ability to conduct current Electroporation The application of high electric field pulses of short duration to create temporary pores holes in the membranes of cells Electroporation Cuvette Square chambers with electrodes on two sides usually measuring 1 mm 2 mm or 4 mm in gap for the purpose of electroporating cells in suspension The cell type being electroporated typically determine the gap Prokaryotic cells typically uses 1 mm or 2 mm gap cuvettes while eukaryotic cells will typically use 2 mm or 4 mm gap sizes Exponential Decay Waveform This waveform is mainly used for transforming cells during electroporation In this type of pulse the set voltage Is released from the capacitor and decays rapidly and exponentially over time millisecs The delivered pulse is characterized by two parameters the field strength kV cm and the time constant These parameters can be adjusted by varying voltage and capacitance settings to achieve a wide pulse gradient Field Strength The potential difference between two points electrodes in Volts divided by the distance between the electrodes called gap and expressed in cm Expressed as V cm or kV cm This is true only if the electric field is homogenous as It is in parallel plate electrodes Gap The distance between electrodes HARVARD APPARATUS BTX Homogenous Electric Field The direction and field strength are constant Hydrostatic Pressure Th
14. up the system by pushing the rocker switch located on the back panel at the bottom left The display will flash the BTX logo Once the software initializes the Home screen will appear You are now ready to begin your work Fuse Holder Main Power Switch Universal Power Input Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com 1 Operating the System Setting Time Date After initializing the home screen will appear Tap the Gears icon HARVARD APPARATUS 02 26 13 9 51 28 AM Tap Time Date icon HARVARD APPARATUS Tap Date icon and use the keypad to type the date Tap Time icon and use the keypad to type the time Tap the Green Check icon to save and return to the settings screen NOTE You may change the date and time format by tapping ya icons to the right of the given values Set Date and Time HARVARD APPARATUS ce i een oojo oojo DGG OOO OO HARVARD APPARATUS Setting Audible Alarm Preferences On the home screen tap the Gears icon HARVARD APPARATUS 02 26 13 9 51 28 AM Tap Audible Alarms icon HARVARD APPARATUS HS Select preferred audible alarms by tapping the icons to activate or deactivate Tap the Green Check icon to save and return to the settings screen AUDIBLE ALARMS HARVARD APPARATUS 06 06 13 16 23 10 O Pulse Delivered D Out of Range O Pulse Aborted O Keypad Clicks Gemin
15. 10 us to 600 us us Multiple Pulsing LV Mode to 10 to 120 10 per sample HV Mode to2 to 36 3 per sample Pulse Interval LV 0 15 to 10S 0 1 sto 10 sec Capacitance in UF LV Mode 37 75 UF 3775 Ur HV Mode GSF 85 UF Voltage Droop 20 Load lt K Q 10 Load gt I KQ 20 Load lt KQ I0 Load gt I K Q Exponential Decay Pulse Ranges LV Mode lO to 500 5 V res 5 to 500 I V res HV Mode 510 to 3000 10 V res 505 to 3000 5 V res Voltage Accuracy LV Mode 5 5 HV Mode 5 5 Time Constant Range LV Mode 1 25 ms to 3 275 S 1 25 ms ms to 5 158 S HV Mode 0 5 ms to 50 ms 0 5 ms 0 5 ms to 133 875 ms Multiple Pulsing Multiple Pulsing No to 12 Rint 100 ohm to 24 Rint gt 100 ohm Pulse Interval N A 5 to 30 sec Capacitance in uF LV Mode 25 to 3275 uF 25 uF res 25 to 3275 uF 25 uF res HV Mode 10 25 50 uF 10 25 35 50 60 75 85 uF Internal Resistance in parallel with load All Modes LV Mode 50 to 1000 Q 50 Q res Sample Resistance Load Exponential Decay or Square Load 10 Q LV 25 to 1575 Q 25 Q res HV 50 tol 575 Q 25 Q res PL lt 00ms load 8 9 Q PL gt 100ms load UU Q HV Mode 510 2500 Load 20 Q 2500 to 3000 V Load 600 Q Other Spcifications Load 40 Q Charging Time LV lt 7 sec HV lt 4 sec LV lt 7 sec HV lt 4 sec Monitoring Puls
16. 15 CHAR MAX Q MOQMDMYOO ASOHOHUWU WOVOQWHO bel L sss OO HARVARD APPARATUS BTX Exponential Decay Wave Tap the Exponential icon Tap the Green Check icon to save HARVARD APPARATUS 02 26 13 3 38 29 PM Square Wave HARVARD APPARATUS 02 26 13 3 39 20 PM MY_NEW_ PROTOCOL o i GAD Co HARVARD APPARATUS 02 26 13 3 40 31 PM Use the numeric keypad to set voltage Tap the Green Check icon to save SET PROTOCOL VOLTAGE vm HARVARD APPARATUS 02 26 13 3 42 48 PM Siate IC o0 Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com 14 Performing Experiments New Protocols continued Exponential Decay Wave continued Tap the Res Cap icon MY_NEW PROTOCOL Gap C 9 7 HARVARD APPARATUS LES tial a 02 26 13 3 45 16 PM Tap the Resistance ohms icon Use the numeric keypad to set resistance value ease eD DAG Bets DICH oo Tap the Capacitance uF icon HARVARD APPARATUS 02 26 13 3 49 48 PM NOTE LV and HV mode capacitance values differ Use the p numeric keypad to set resistance value SET RES AND CAP Gao Mao olojo HARVARD APPARATUS 02 26 13 3 53 54 PM DG LOL OO HARVARD APPARATUS Tap the Pulses Interval icon Tap the Pulses icon Use the numeric keypad to set the of pulses NOTE Only the BTX Gemini X2 can perform multiple pulsing with Exponential Dec
17. 9 PM Voltage v 100 102 Pulses delivered 1 Duration ms 191 Resistance ohm Droop DIT Plate PRESS TO CLEAR MESSAGE of Pulses 1 Handler Pulse Interval s Gap width mm 4 Meas Load ohm OK OO Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com 20 HARVARD APPARATUS Performing Experiments Customizing a Preset Protocol continued Scroll through user protocols to locate the newly saved method Use keyboard to name copy of protocol Double tap the newly saved method Tap the Green Check icon to save HARVARD APPARATUS Copy To 02 26 13 2 54 25 PM User Protocol HEK_293_CUSTOM_ 15 CHAR MAX DWED ASOHOHYWW BWOMOMMO Tap the parameter s requiring customization The message board will turn green and alert the user that the Proceed to modify selected parameters method has been copied Tap the Green Check icon to proceed to the run screen Tap the Home icon HEK_293 CUSTOM GAP C 3 3 HARVARD APPARATUS HARVARD AFPARATUS 02 26 13 3 26 47 PM 02 26 13 2 46 20 PM Using an HT Plate Handler BTX Gemini X Only Plug the HT Plate Handler into the front ports of the HARVARD APPARATUS BTX Gemini X2 Preset Protocols E Scroll through available cells Double tap desired cells User Protocols PROTOCOL SELECT HARVARD APPARATUS 22 Protocols 02 27 13 8 59 51 AM 59 51 AM e Ss rt stem cells CHO Gemini
18. 99 e www btxonline com 32 HARVARD APPARATUS Upgrading BIX Gemini Series Software USB Virtual Commport Driver Installation When you connect the BTX Gemini X to a computer via USB for the first time Windows will seek to install a driver for communication The following section details the installation of the Virtual CommPort Driver supplied with the BTX Gemini X 1 Connect the Gemini X generator to the computer via USB Open the control panel and select System and Security and then System Click Device Manager on the left hand menu Mating Face Pin Signal 1 5V 2 Data 3 Data a gt 7 ee E E 7 4 H J gt Content Panel System and Security System Ne Camere Panet Hame View basic information about your computer X Device Manager Windows ellen Remote settings agora 7 Profeconnal Sytem protection Capynghe Z 2009 Microsoft Comorian Al nghts reserved X Advanced iystern settings Serg Pack 1 Get more features with new edition of Windows 7 2 Two Flash update applications will be displayed in the device manager under Other Devices Right click on either of the flash update applications and select Update Driver Software e zl IRerlelzgg b lt 2 Keyboards gt D Mice and other pointing devices b a Network adapters 4 7 Other devices fi Device a 7 Ports COM amp LI 7 Intel R Activ D Processors wx Sound video an Q Segre fe System devices a Universal
19. Operate with Suspected Failures If damage is suspected on or to the product do not operate the product Contact qualified service personnel to perform inspection Orient the Equipment Properly Do not orient the equipment so that It is difficult to operate the disconnection device Place Product in Proper Environment Review the operating manual for guidelines for proper operating environments Do Not Operate in Low Impedance Sample or Load If the electroporation samples have an impedance of less than 20 Q in LV and 40 Q in HV this may result in sample loss and potential damage to the unit Observe All Warning Labels on Product Read all labels on product to ensure proper usage Caution Protective Ground Terminal Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com 4 Caution Risk of Electric Shock HARVARD APPARATUS BTX HARVARD APPARATUS Generator Specifications Generator Specifications V7 BIS Gemini SC BTX Gemini X Square Wave Pulse Ranges Voltage Range Voltage Range LV Mode 10 to 500 5 V res 5 to 500 I V res HV Mode 510 to 3000 10 V res 505 to 3000 5 V res Voltage Accuracy LV Mode 5 5 HV Mode 5 5 Pulse Length Range LV Mode 50 us to 10 ms 50 us res 10 ms to 100 ms ms res 1O us to 999 us us res ms to 999 ms ms res HV Mode 50 us to 5 ms 50 Ls res
20. PARATUS Installed Protocols he Electroporation Experts Download Protocols m Protocol Select From PC To Unit Upload Protocols From Unit To PC COM Port F Yas_smooth_musc COM5 e COM Port List Retrest Protocol ma Cor BT Send Unit Commands man Step Step 0 Manage Log Files Pulse Data Cancel Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com 30 HARVARD APPARATUS BTX Protocol Manager Sending Commands to Generator Upload Log Files 1 Click Command 1 On the main screen click Pulse Data 2 Type desired command 2 Select your desired files or folders and then click Transfer 3 Click Send NOTE Files should be backed up periodically The generator s By NOTE If unexpected responses are seen type echo off SD card can store up to approximately 100 000 files Protocol Data Experimental Data Files Day Subdirectory Selected ear Month Log Files 164230 TXT 164305 TXT 164321 TXT 164415 TXT 164432 TT 164458 TXT 164509 TXT 164657 TXT 164730 TXT gt lt Delete Transfer Cancel 3 Select the drive or folder location Troubleshooting Protocol Manager 1 Do not upload download Protocols while generator is running Program works best while generator is idle on Protocol setup screen 2 Order of Operations A Connect USB B Turn gener
21. Petri Dish Tissue Chamber 15 x 15 mm Ea AP In Vivo IM needs 45 0216 47 0086 6 Needle Array 6 mm gap 16 mm Length Ea 45 0507 Petri Dish Tissue Chamber 15 x 15 mm Kit Ea AP In Vivo IM 45 0100 Petri Dish Electrode 2 mm gap 90 mm Well Dish Kit Ea 47 0204N 5 mi Chamber for Agile Pulse Max Ea 45 0130 Petri Pulser for 6 Well Plates 35 mm Well Kit Ea 47 0090 Electrode Adapter Box for Agile Pulse In Vivo Ea 45 0490 Tissue Slice Chamber 7 x 7 mm Kit Ea 47 0206 10 ml Chamber for Agile Pulse Max Ea Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com Declaration of Conformity Manufacturer Harvard Apparatus Inc 84 October Hill Road Holliston Massachusetts 01746 1388 U S A Phone 508 893 8999 We herewith declare that the following product Product Name Gemini X Gemini SC To which this declaration relates is in conformity with the applicable EC Directives harmonized standards and other normative requirements Application of Council Directive s 2006 95 EC Low Voltage Directive 2004 108 EC Electromagnetic Compatibility Directive RoHS Compliant Standard s to which conformity is declared Safety EN 61010 1 2012 3rd Edition Emmissions Immunity EN 61326 2006 05 01 FR47 FCC Part I5 Sub Part B 2013 EN 61000
22. RATUS 06 06 13 16 27 19 Rename Delete Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com 28 Protocol Manager Installation Insert the generator CD into the CDROM a file containing the CD s contents will open automatically Navigate to the Protol Manager folder under Applications Right click on Protocol Manager Select Send To then Desktop to create a shortcut on your desktop By NOTE Compatible with Windows XP amp Windows 7 1 Connect the generator to the PC using a USB cable 2 Turn generator on 3 Start Protocol Manager 4 Select the proper COM port Overview Protocol Manager allows the user to e Upload protocols from the Gemini X generator to a PC e Download protocols from PC to the Gemini X generator e Send commands to the Gemini X generator using a PC e Upload log files from the Gemini X generator to PC 2 Protocol Manager File Help Advanced Protocol Manager ARVARD APPARATUS Protocol Select Download Protocols From PC To Unit Installed Protocols Upload Protocols From Unit To PC COM Port COMS D COM Pott List Refresh Send Unit Commands Command Manage Log Files Pulse Data Log File Control Choose Protocol PC to Generator Protocol Control Refresh Delete Save Protocol Generator to PC Protocol Control HARVARD APPARATUS BTX Up
23. RD APPARATUS Voltage 50000_ 04 03 13 11 06 19 AM DAC DOG oS OV Power Failure Notification If power is interrupted during operation an alarm will sound to alert the user to the interruption When power is restored an onscreen message is displayed to indicate the interruption MAIN MENU HARVARD APPARATUS 02 26 13 4 19 11 AM User Protocols Low Battery Indication Time Date area will turn red when battery is low SET DISPLAY BRIGHTNESS HARVARD APPARATUS Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com 36 HARVARD APPARATUS BTX As described electroporation Is the application of controlled direct current DC electrical pulses which are applied to living cells and tissues for a short duration of time The pulse induces a transmembrane potential which causes the reversible breakdown of the cellular membrane This action results in the permeation or pore formation of the cell membrane which allows small molecules such as dye oligonucleotides or peptides and large molecules such as proteins DNA and RNA to be introduced into the cell During this process the cellular uptake of the molecules continue until the pores close which can take milliseconds to minutes Optimization of the electroporation process involves several factors Choosing the wave form determining field strength and adjusting pulse length are just a few critical
24. Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com EA Performing Experiments Using an HT Plate Handler BTX Gemini X Only continued Review parameters Tap Plate Handler icon RUN PROTOCOL HARVARD APPARATUS HEK 293 867 EE Square Wave Set Meas 04 02 13 4 16 48 PM Voltage Y 100 Duration ms 10 Resistance ohm Droop Plate of Pulses i Handler Pulse Interval s Gap width mm 4 Meas Load ohm Using the numeric keypad icon type the number of columns being pulsed Tap the Green Check icon to save HARVARD APPARATUS 02 27 13 9 00 18 AM GIS lt NOTE Note the message box It will indicate how many ZB pulses should be set in the HT plate handler This number will be a combination of both pre pulse resistance measurements and DC pulses SET PLATE COLUMNS HARVARD APPARATUS Columns 02 27 13 9 00 41 AM LS 2 0 OO HARVARD APPARATUS BTX Review parameters Set the HT plate handler with the correct number of pre pulse resistance measurement and DC pulses Tap the Go icon to run protocol SET PLATE COLUMNS HARVARD APPARATUS HEK 293 867 a IEE SE pes Square Wave Set Meas 04 02 13 4 21 40 PM Voltage v 100 Duration ms 10 Resistance ohm Droop Plate of Pulses 1 Handler Pulse Interval s Gap width mm 4 Meas Load ohm t The Square icon can be used to stop the p
25. _293 UU HEK_293_CUSTOM O Hela 1 pu zen rporocofll L C 4 Use the numeric keypad to set the pulse length Tap the Green Check icon to save Tap the Voltage icon HARVARD APPARATUS 02 26 13 4 26 39 PM MY Ke GAD C9 7 HARVARD APPARATUS HARVARD APPARATUS 02 26 13 4 20 34 PM DG S L COLL o0 GED gt or Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com 17 HARVARD APPARATUS Performing Experiments New Protocols continued Tap the Gap icon d Square Wave continued ir MEN PROTOCOL Gap C 9 7 HARVARD APPARATUS L D L ave h Tap the Pulses Interval icon 02 26 13 4 30 09 PM My MEN PROTOCOL V NEW PR GAP C 0 HARVARD APPARATUS 02 26 13 4 28 20 PM HR Use the numeric keypad to set the gap between electrodes Tap the Green Check icon to save Tap the Pulses icon HARVARD APPARATUS Use the numeric keypad to set the of pulses 02 26 13 4 33 37 PM SET PULSES AND INTERVAL HARVARD APPARATUS 02 26 13 4 29 28 PM Mee Tap the Interval sec icon Use the numeric keypad to set the pulse interval Tap the Green Check icon to save SET PULSES AND INTERVAL HARVARD APPARATUS 02 26 13 4 29 57 PM nterval Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com 18 HARVARD APPARATUS Performing Experiments New Protocols conti
26. able numbers and symbols that can be included in the Protocol Name To switch the keyboard back to letters mode choose the Letters button ABC 15 characters maximum 5 When finished entering the new Protocol Name choose Accept to save your changes and return to the File Options screen 6 Choose BACK to return to the Mode Selection screen Managing Gemini X Series Protocols HARVARD APPARATUS BIX Renaming a Protocol Follow these instructions to rename a previously saved user protocol 8 From the Protocol Configuration screen choose the Protocol Name button to enter the Protocol Mode screen From the list of available Protocols select press once the Protocol you want to rename then choose the File Options button to enter the File Options screen Choose Rename Protocol to access the Protocol Name Entry screen The current Protocol Name will be displayed in the text entry box On the Protocol Name screen type the new name for your Protocol using the onscreen keyboard Choose the Symbols Numbers button 123 to display the list of available numbers and symbols that can be included in the Protocol Name To switch the keyboard back to letters mode choose the Letters button ABC 15 characters maximum When finished entering the new Protocol Name choose Accept to save your changes and return to the File Options screen Choose Back to return to the Protocol OPTIONS FOR PROTOCOL 1 HARVARD APPA
27. aiorano N A et al Promotion of embryonic cortico cerebral neuronogenesis by miR 124 Neural Development 4 40 2009 Ex Vivo Electroporation Deora A A et al Efficient Electroporation of DNA and Protein into Confluent and Differentiated Epithelial Cells in Culture Traffic 8 1304 1312 2007 Thomas J L et al Electroporation an alternative to biolostics for transfection of Bombyx mori embryos and larval tissues Journal of Insect Science 3 17 2003 Dimitrov D S and Sowers A E 1990 Membrane electroporation fast molecular exchange by electroosmosis Biochimica et Biophysica Acta 1022 381 392 Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com Applications General References continued Ex Vivo Electroporation continued Deora A A et al Efficient Electroporation of DNA and Protein into Confluent and Differentiated Epithelial Cells in Culture Traffic 8 1304 1312 2007 Thomas J L et al Electroporation an alternative to biolostics for transfection of Bombyx mori embryos and larval tissues Journal of Insect Science 3 17 2003 Dimitrov D S and Sowers A E 1990 Membrane electroporation fast molecular exchange by electroosmosis Biochimica et Biophysica Acta 1022 381 392 Sukharev SI Klenchin VA Serov SM Chernomordik LV and Chizmadzhev YA 1992 Electroporation and electrophoretic DNA transfer into cells The eff
28. ame high quality researchers have come to expect from Harvard Apparatus BIX What is the difference between the Gemini X2 and the Gemini SC2 The Gemini X is designed to give researchers the ultimate control and flexibility in their experiments by making it possible to perform electroporation on tissues and organs in vivo as well as in utero in ovo ex plant on adherent cells as well as cells in suspension in either single cuvettes or 96 well plates can be controlled remotely via footswitch or PC and it offers storage of pulse data Because of the Gemini X2 broad range of use the specifications are wide ranging making the Gemini X2 the most versatile electroporation system available today The Gemini SC is designed for researchers working to electroporate cells in suspension in cuvettes This system cannot accommodate multiple pulsing with the exponential decay waveform remote operation specialty electrodes or 96 well options For this reason some of the specifications of the Gemini SC are not as extensive as what is available in the Gemini X2 Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com HARVARD APPARATUS Unpacking the System The shipping carton in which your BTX Gemini Twin Waveform Touchscreen Display Electroporation System Is packed has been specifically designed to provide maximum protection to the instrument during transportation and norma
29. an be electrically charged exhibits capacitance Capacitor A device that stores energy in the form of an electric field A capacitor consists of two metal plates insulated from each other by a dielectric insulating usually a plastic material such as Mylar material In an ideal capacitor no conduction current flows between the plates after the capacitor is completely charged Capacitors can be fixed variable or adjustable Cell Form The format in which cells present for experimentation Cell forms include suspension in vivo in ovo ex plant adherent whole organism etc Dielectric Breakdown The reversible breakdown of bi lipid layer membranes as a result of the application of a DC electroporation pulse A sufficiently high field strength may increase the membrane potential past a critical point leading to the breakdown of the membrane Dielectric Constant For a given dielectric nonmetallic material the ratio of electrical capacitance of a dielectric filled capacitor to a vacuum capacitor of identical dimensions Divergence The deviation of field lines e g electric field lines from parallel homogeneous conditions A highly divergent field is a very inhomogeneous field where the value and direction of the field change drastically in the area under consideration Electrolytic A fluid containing charged molecules is called an electrolyte Electrolytic properties are associated with such a fluid such as
30. ator on C Start Protocol Manager program Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com 3 Maintenance The BTX Gemini Twin Wave Series generators require no special maintenance other than keeping them clean To clean the exterior surfaces use a lint free cloth to remove loose dust Use care to avoid scratching the clear display window For more efficient cleaning use a soft cloth dampened not soaked with an aqueous solution of 75 isopropyl alcohol or a mild detergent Fuse Replacement Key Information 1 Make sure the power cord is disconnected from the main supply before servicing the fuse 2 Use only Type 3AG 1 4 x 11 4 in 5 amp 250 volt Type T time delay fuses Turn off power and remove power cord from power module Use a straight blade screw driver to pry open the access door Remove the fuse holder and then remove the fuses from this holder as shown in figure below Replace fuses and then replace the fuse holder HARVARD APPARATUS BTX Battery Replacement 1 Remove screw and swing cover to the side 2 Slide battery out from under clip 3 Install Lithium Coin Battery 3V 16mm CR1620 by sliding under clip with positive side facing out CAUTION Use only same type and rated battery Observe ya polarity when installing Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 89
31. ay Wave the BTX Gemini SC cannot perform multiple pulsing with Exponential decay waves SET PULSES AND INTERVAL HARVARD APPARATUS 02 26 13 3 59 14 PM oojo Tap the Interval sec icon Use the numeric keypad to set the interval between pulses Tap the Green Check icon to save NOTE Intervals must only be set when performing p multiple pulsing SET PULSES AND INTERVAL HARVARD APPARATUS Grus JE 02 26 13 4 00 12 PM 7 Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com HARVARD APPARATUS BTX Performing Experiments New Protocols continued View your protocol Exponential Decay Wave continued To run tap the Omega icon Tap the Gap icon RUN PROTOCOL HARVARD APPARATUS MY_NEW_PROTOCOL LE AE E oe eee ee Exponential Decay Set Meas 04 02 13 3 29 11 PM Voltage v 3000 Time Const ms 5 Resistance ohm 200 Capacitance uF 25 Plate MY_NEW PROTOCOL Gap Co HARVARD APPARATUS 02 26 13 3 45 16 PM of Pulses 1 Handler Pulse Interval s Gap width mm 2 Meas Load ohm TC t With load measurement OK press the Go icon to run protocol Use the numeric keypad to set the gap between electrodes RUN PROTOCOL HARVARD APPARATUS NY NEW PROTOCOL eee Tap the Green Check icon to save Exponential Decay Get Meas 04 02 13 3 31 23 PM HARVARD APPARATUS 02 26 13 4 03 56 PM Voltage v 3000 Time Const ms 4 18 Po Resis
32. ay list Scroll Up Used to scroll up in a display list Settings Access the settings menu used to adjust the following parameters Date and Time Audible Alarms Backlighting and software updates Displays device information OU OHO HARVARD APPARATUS BTX Stop Protocol Used during the pulse delivery sequence to stop the progress of the protocol Square Wave Indicator Used to indicate when a protocol utilizes square wave pulses Touch Screen Locked Indicates that the touch screen is currently locked Press the icon and enter password to unlock the touch screen Touch Screen Unlocked Indicates that the touch screen is currently unlocked Pressing the icon twice will allow the user to password protect the protocol Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com Display Error Message Power Loss Error Error Messages amp Troubleshooting Error Description Power loss during pulsing HARVARD APPARATUS User Troubleshooting Power lost while protocol was runing PFC Over Temp Pfc module over temperature Hardware error Please contact the manufacturer Interlock Open Electrode interlock open Connect electrode connectors to the front of the generator and make sure the cuvett safety dome cover is closed IGBT Switch Fault IGBT switches fault Hardware error Please contact the manufacturer Charg
33. e Voltage Width Droop and Sample Resistance Pulse Voltage Width Droop and Sample Resistance Display 4 3 in color display 4 3 in color display Controls Touch Screen Touch Screen Programmability Storage over 1000 Protocols Storage over 000 Protocols Arc Control Yes Yes When used in Plate Handler mode Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com D Generator Specifications BTX Gemini SC2 BTX Gemini X2 Generator Specifications V7 Other Spcifications continued Interlock Switches Yes Yes Pre Pulse Sample resistance Yes Yes check Pulse Over Current Protection Yes Yes PC control No Yes PC communications No Yes Log Report No Yes Remote Operation No Yes Foot Switch Control No Yes Power Ratings 35 W idle and 350 W pulsing 35W idle and 350 W pulsing Input Voltage ratings 100 to 240 VAC 100 to 240 VAC CE ETL Marking CB Scheme Yes Yes EuRoHS Dimensions H x W x D inches 12 75 x 11 25 x 8 5 KC H Weight 6 b 16 b Warranty year 2 years Atmospheric Specifications Operating Temperature 4 C to 40 C 40 F to 104 F Storage Temperature 10 C to 70 C 14 F to 158 F Operating Humidity See Chart Below Storage Humidity 20 to 80 RH non condensing Mode of Operation Continuous Classification
34. e Voltage Max Error Charging voltage above the expected voltage Turn off the unit for a few minutes then turn back on Re run the protocol if the issue recurs contact the manufacturer Charge Current Max Error Charging current above the expected current Turn off the unit for a few minutes then turn back on Re run the protocol if the issue recurs contact the manufacturer LV Cap Bank Max Low voltage capacitor bank voltage above expected value Turn off the unit for a few minutes then turn back on Re run the protocol if the issue recurs contact the manufacturer LV Cap Bank Min Low voltage capacitor bank voltage below expected value Turn off the unit for a few minutes then turn back on Re run the protocol if the issue recurs contact the manufacturer HV Cap Bank Max High voltage capacitor bank voltage above expected value Turn off the unit for a few minutes then turn back on Re run the protocol if the issue recurs contact the manufacturer HV Cap Bank Min High voltage capacitor bank voltage below expected value Turn off the unit for a few minutes then turn back on Re run the protocol if the issue recurs contact the manufacturer Charge Time Max Charging capacitor bank to selected voltage exceeded max time estimated Turn off the unit for a few minutes then turn back on Re run the protocol if the issue recurs contact the manufacturer
35. e pressure in liquids at rest Inhomogeneous Electric Field Direction and strength of the electric field vary Number of Pulses The number of pulses the sample will be exposed to Osmotic Pressure The applied pressure required to prevent the flow of solvents of different concentration across a semi permeable membrane Pore A small mostly transient opening in a cell wall caused by the application of a brief high electric field pulse Potential Difference The difference in Volts between points in an area between electrodes Protocols The method for performing an experiment Pressure Gradient The difference in pressure between two points in a medium Pulse Interval The time between multiple pulses Pulse Length Pulse Duration The length of time an electric signal is applied Specialty Electrodes Electrodes used with the BTX Gemini X2 to perform electroporation on a wide variety of cell forms Some applications include in vivo in ovo in utero or ex plant electroporation Harvard Apparatus BTX offer many types of specialty electrodes Square Waveform This waveform is typically used for eukaryotic cells It is is characterized by the voltage delivered the duration of the pulse the number of pulses and the length of the interval between pulses Time Constant represented by the greek letter tau T is the amount of time required for the actual voltage of the delivered pulse
36. ear protein and putative tumor suppressor expressed during Myeloid Cell differentiation Stem Cells 22 832 848 2004 JonathanM Quinlan Wei Yuan Yu MarkA Hornsey David Tosh andJonathan MWsSlack In vitro culture ofembryonic mouse intestinal epithelium cell differen tiation and introduction or reporter genes BMC Developmental Biology 6 24 2006 YangbingZhao ZhiliZheng Cyrille J Cohen Luca Gattinoni Douglas C Palmer Nicholas P Restifo Steven A Rosenberg and RichardA Morgan High efficiency transfection ofprimary human and mouse Tlymphocytes using RNA electropora tion Molecular Therapy 2006 13 151 159 HARVARD APPARATUS BTX William J Buchser Jose R Pardinas Yan Shi John L Bixby and VanceP Lemmon 96 Well electroporation methodfor transfection of mammalian central neurons BioTechniques Vol 41 no 5 2006 K Regha AjitK Satapathy and Malay K Ray RecD plays an essentialfunction during growth at low temperature in the Antarctic Bacterium Pseudomonas syringae Lz4W Genetics 170 1473 1484 August2005 Victor B Busov R Meilan D W Pearce C Ma S B Rood and H Strauss Activation Tagging ofa dominanat Gibberellin catabolism gene GA 2 oxidase from poplar that regulates tree stature Plant Physiology Vol 132 pp 1283 1291 July 2003 Jun Ishikawa Kazuhiro Chiba Haruyo Kurita and Hiroyuki Satoh Contribution of rooB2 RNA Polymerase Subunit Gene to Rifampin Resistance in Nocardia Species An
37. ect of DNA interaction with electropores 1992 Biophys J 63 1320 1327 Nickoloff Jac A ed 1995 Plant Cell Electroporation and Flectrofusion Protocols Methods in Molecular Biology Volume 55 Humana Press Totowa New Jersey E A Disalvo and S A Simon eds 1995 Permeability and Stability of Lipid Bilayers CRC Press Boca Raton p 105 121 Chang D C Chassy B M Saunders J A and Sowers A E eds 1992 Guide to Electroporation and Electrofusion Academic press San Diego 581 pp Neuman E Sowers A E and Jordan C A eds 1989 Flectroporation and Electrofusion in Cell Biology Plenum Press New York 587 pp Bartoletti D C Harrison G I amp Weaver J C 1989 The number of molecules taken up by electroporated cells quantitative determination FEBS Lett 256 4 10 Djuzenova C S Zimmermann U Frank H Sukhorukov V L Richter E amp Fuhr G 1996 Effect of medium conductivity and composition on the uptake of propidium iodide into electropermeabilized myeloma cells Biochim Biophys Acta 1284 143 152 Klenchin VA Sukharev SM Chernomordik LV Chizmadzhev YA Flectricaly induced DNA uptake by cells is a fast process involving DNA electrophoresis 1991 Biophys J 60 804 811 Neumann E Kakorin S amp Toensing K 1999 Fundamentals of electroporative delivery of drugs and genes Bioelectrochem Bioenerg 48 3 16 Neuman E Toensing K Kakorin S Budde P
38. ectroporation Discussion Electroporation is the use of a transmembrane electric field pulse to induce microscopic pathways pores in a bio membrane Their presence allows molecules ions and water to pass from one side of the membrane to the other When the electric field is applied the ions inside and outside the cell membrane migrate As the charge builds up on either side of the membrane the membrane weakens and the pathways form permitting material outside of the cell to enter If the electric field is promptly removed the pathways close and the membrane reseals If the electric field duration is too long the pathways increase and the cell is killed Efficient electroporation depends on proper selection of electric field waveforms The electropores are located primarily on the membrane areas which are closest to the electrodes The pathways form in about a microsecond and seal in seconds to minutes The duration of the electric field is tens of microseconds to tens of milliseconds The use of electroporation was described by Neumann in the early 1980 s The routine use of electroporation became very popular with researchers through the 1980 s because it was found to be a practical way to place drugs or other molecules into cells In the late 1980 s scientists began to use electroporation for applications in multi cellular tissue In the early 1990 s Lluis Mir of the Institute Gustave Roussy was the first to use electroporation in a human t
39. eeeeeeeeeeeseseeseeeeteeeeeeeeeeeeeeeeeeeeeeeeeeeeesssaaeaaaaaaaeeeesees 12 Ee el 13 Eis Ole Vii A Seen ile COI E 13 Performing EXDEriMent guest ege geed EES SEEAEREEEEEEEENE REESEN i nii 14 25 KEN TH 14 19 PPS aa ee ee 19 20 Ree NEE Protocol E 20 21 Kuer EE EE 21 23 Re a e eee T 24 eS SON PIO SG ING FO E tnteanos sauce ereceeneoauee 25 Touchscreen Button Reference ENNEN 26 Error Messages amp Troubleshooting esse sees sees eee 27 Managing Gemini X Series Protocols css sees eee eee eee 28 Protocol I AC TH 29 31 DEE 29 KEE 29 MIG eles GS ee 6 EE T E EE teen teeniatenandaaacucet A EI E ETTE E 29 Download PC to re 30 Se neen Eelere EN ee RE EN PUT AICS E E 32 Upgrading BTX Gemini Series Software NENNEN 33 35 USB Virtual Commport Driver 1nstallation sss css sss sss ssc cs sss scoe sc esen neee ee ennenen an gonnnar 33 34 RA HI ER a Calen T 35 gef ET EE 36 General Optimization Guide for Electroporation EE 37 38 Wave 9 E 37 SI en EE FS D E ER 38 Number OT E 38 ee 38 DNA RNA get ER ele 39 41 Mammahan CONT Aa e Oa EEN 39 In Vivo In Utero IN e 39 Bacteria and Yeast Transformation tnr sees seen H HHO t ttEEEEEEEEEEAAEAEE EErEE rEEEEEEEEt 39 Plant and Insect TranstectiON saogz zaot oZ E 92994 cnz aana EE EEEE EEEE NEEE 39 BTT 39 ENER 39 41 Electroporation Buffers siicidsciccecicssaictmasatmsasacnesasattnesisacnstacstiasastacsstacaniecaxtecansncausesaxeddanecaixesaixanasxs 42 Experiment Troubleshooting ss sssszssgas
40. ellular uptake and expression of DNA siRNA and miRNA in muscle skin liver kidney testis retina tumors etc In vivo electroporation has successfully been used in embryo applications in utero and in ovo applications in addition to transfection of Zebra fish Bacteria and Yeast Transformation Electroporation has long been recognized as the most efficient means of transforming both gram negative and gram positive bacteria and yeast Gramnegative bacteria such as coli or Helicobacter pylori are generally easier to transform than grampositive bacteria e g Streptococcus pneumoniae due to their cell wall composition Transformation efficiencies of 1x10 transformants ug DNA are commonly seen for gram negative bacteria while for gram positive bacteria generally 1x10 transformants ug DNA are achievable Plant and Insect Transfection Electroporation of plant tissue can be used to generate transgenic crops that are useful in agricultural horticultural applications Insect models are also widely used throughout the scientific community to study development and gene regulation and function The ability to introduce genes or molecules is essential to researchers working with either of these two species This is why researchers consistently turn to Harvard Apparatus BTX for all of their electroporation needs References Jonathan M Dermott J M Gooya B Asefa S R Weiler M Smith J R Keller Inhibition of Growth by p205 A nucl
41. ems HARVARD APPARATUS BTX These instruments contain a high voltage power supply adjustable to 3 000 V High voltage power supplies present a serious risk of personal injury if not used in accordance with design and or use specifications if used in applications on products for which they are not intended or designed or if they are used by untrained or unqualified personnel e The user must read this manual carefully before the instruments are placed into operation e Removing the cover will void the warranty e Do not connect or disconnect the high voltage cable with the high voltage enabled e To connect or disconnect the cable turn line power off and unplug line mains cord e Do not touch the electrode tip while the waveforms are being applied If there are any questions about the operation of this instrument call Harvard Apparatus BTX Customer service at 1 800 272 2775 or 1 508 893 8999 Out of Warranty Service Proceed exactly as for Warranty Service above If our service department can assist you by phone or other correspondence we will be glad to help at no charge Repair service will be billed on the basis of labor and materials A complete statement of time spent and materials used will be supplied Shipment to Harvard Apparatus BTX should be prepaid Your bill will include return shipment freight charges Disassembly by the user is prohibited Service should only be carried out by experienced Harvard Apparat
42. es 610 45 2020 BTX Safety Dome for SC2 lea G SE Gula neas 45 2021 BTX Safety Dome for X ea 45 2006 BTX Gemini X Generator only ea its See LS E 45 2007 BTX Gemini X2 Electroporation System ea E E SE Includes Gemini X2 Generator Cuvettes 610 620 45 0465 HT 25 Well Adapter Plate ea 640 pack of 30 10 ea Safety Dome 2 45 0468 Plate Handler Pins pk 25 25 ea and 660 Cuvette Rack 45 0469 Plate Handler Pins pk 100 100 ea 45 2008 Ge SE er ea 5012 017 Pliers for Plate Handler Removing Pins ea 640 pack of 30 10 ea Safety Dome 2 HT 200 4500 012 25 Well Plate Seal 2 ea Plate Handler x 2 mm gap HT Plate x 4mm 4500 015 96 Well Plate Seal Je Cuvettes 45 0216 Connection Cable Cable 0 ft Banana to ea 45 0124 Cuvette Plus mm gap 90 ul pk 50 Gray 50 pk Micrograbber 45 0125 Cuvette Plus 2 mm gap 400 ul pk 50 Blue 50 pk 45 0204 Tweezertrode Cable Single Adaptor Cable for ea 45 0126 Cuvette Plus 4 mm gap 800 UT pk 50 Yellow 50 pk See See Ode ae 45 0140 Bulk Cuvette mm 24 pk 100 Gray Case 2400 ea 45 0503 pane eee fe ue A slice ea 45 0141 Bulk Cuvette 2 mm gap 24 pk I00 Blue Case 2400 ea 45 0142 Bulk Cuvette 4 mm gap 24 pk I00 Yellow Case 2400 ea eee E E 45 2031 BTX USB Cable 2 m ea EE 45 2032 BTX USB Cable 5 m ea ease A eae ee oe SS 45 0217 Electrode Cable for Flat Electrode 10 ft BTB ea 45 0463 25 Well Plate 4 mm gap 250 ul pk 6 6 pk 45 0088 Banana Splice F F Adapter Se
43. es with longer pulse lengths Buffers such as EDTA or Tris can drastically reduce the transfection efficiency Therefore we recommend resuspending DNA in distilled water Finally electroporating ligation mixtures into E coli can cause arcing and reduced transformations Diluting the ligation mixture a minimum of 1 5 with diH2O dialysis or ethanol precipitation can significantly improve transformation efficiencies and reduce the potential for arcing E Viability Points Light Emission Meaured 20 25 30 35 40 45 Field Strength Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com Applications Mammalian Cell Transfection Electroporation is a highly flexible technique used to genetically modify mammalian cells Whether you are studying up or down regulation of genes specific protein expression This method is non toxic and requires no expensive reagents to successfully transfect your cells Primary cells stem cells or established cell lines can be electroporated with yield high transfection efficiencies and great cell survival rates In Vivo In Utero In Ovo Square waves system allow researchers to set the pulse lengths and number of pulses which is critical to ensure viable cells and tissues while still maintaining efficient transfection both in vivo and ex vivo Electroporation mediated gene and drug delivery has been shown to substantially increase intrac
44. et Meas 94 03 13 10 40 10 AM Voltage v 35 33 9 Pulses delivered 5 Duration ms SORES Resistance ohm Droop T Plate PRESS TO CLEAR MESSAGE of Pulses 5 Handler Pulse Interval s 1 0 Gap width mm 4 Meas Load ohm OK DH 60 Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com 23 HARVARD APPARATUS Performing Experiments Deleting a Protocol Confirm delete by tapping the Delete icon again HARVARD APPARATUS 02 26 13 4 47 15 PM HARVARD APPARATUS 02 26 13 4 49 40 PM Tap the protocol you wish to delete Tap Folder icon HARVARD APPARATUS 02 26 13 4 48 00 PM Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com 24 Performing Experiments Password Protecting a Protocol On the home screen tap the User Protocols icon HARVARD APPARATUS 02 26 13 4 47 15 PM Double tap protocol you wish to password protect HARVARD APPARATUS 02 26 13 5 10 11 PM View the protocol Tap the Open Lock icon PEK 293 STET GAPL 2 7 HARVARD APPARATUS 02 26 13 5 10 26 PM 10 26 PM L goo at ie Pulses Interval HARVARD APPARATUS Confirm password protection by tapping the Open Lock Icon again HEK_293_CUSTOM a GAP C 3 1 HARVARD APPARATUS 02 26 13 5 10 39 PM Press the lock button again to lock the screen Use the numeric keypad to
45. i Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com Operating the System Setting Backlight Preferences On the home screen tap the Gears icon HARVARD APPARATUS 02 26 13 9 51 28 AM Tap Backlight icon SETTINGS HARVARD APPARATUS oes Select preferred brightness by tapping the icons indicating brightness Tap the Green Check icon to save and return to the settings screen SET DISPLAY BRIGHTNESS HARVARD APPARATUS 04 03 13 11 34 54 AM HARVARD APPARATUS Displaying Device Information On the home screen tap the Gears icon HARVARD APPARATUS 02 26 13 9 51 28 AM Tap Device Information icon SETTINGS HARVARD APPARATUS oe 00 View device information Tap the Green Check icon to return to the settings screen UNIT INFORMATION HARVARD APPARATUS Model 02 26 13 12 08 35 PM BTX GEMINI X2 Software Version 3 0 0 2 0 2 24 Protocol Memory Available 1010 Protocols Serial Number G221001 Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com Performing Experiments New Protocols On the home screen tap User Protocols icon HARVARD APPARATUS 02 26 13 9 51 28 AM Double tap New Protocol HARVARD APPARATUS men EE SES HEK_293 Hela 1 TEST Use keyboard to name protocol Tap the Green Check icon to save Erotoco MY_NEW_PROTOCOL
46. iency of the electroporation for several reasons The majority of mammalian cell lines are effectively electroporated at room temperature Samples which are pulsed at high voltage or exposed to multiple pulses and long pulse durations can cause the sample to heat up These conditions cause increased cell death and lowers the transfection efficiency Maintaining the sample at lower temperatures can diminish the heating effects on cell viability and efficiency Since electroporation causes the transient formation of pores keeping the cells at a lower temperature following the pulse may allow the pores to remain open longer to allow more uptake of the exogenous molecules Yet lower temperatures on other cell lines can be damaging and cause high cell mortality This effect is specific to each cell line and should be considered during optimization studies The standard pulse voltage used for cells at room temperature will need to be approximately doubled for electroporation at 4 C in order to effectively permeate the cell membrane Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com Pulse Length The pulse length is the duration of time the sample is exposed to the pulse This is measured as time in micro to milliseconds ranges Adjusting this parameter is dependent on the pulse wave form The pulse length in a square wave system can be inputted directly The pulse length in an ex
47. l handling conditions Upon receipt the carton should be examined for any external damage resulting from shipment Open the carton and carefully remove the BTX Gemini Twin Waveform Electroporator and inspect the unit for any apparent damage Save the carton and packing materials for future transportation and shipping requirements Packing Data Check the packing slip to ensure that all items ordered and listed are included in the shipment Inform Harvard Apparatus BTX immediately if any parts are missing or damaged Power Source As received the instrument is ready for use with either 100 240 V AC 50 60 HZ The power requirements are 350 watts In the USA the power USB Serial Input Footswitch Input High Voltage Output cord has a standard three prong plug switch sold separately Installation Once you have determined that the components of the system have not sustained any obvious damage in shipment proceed with the installation The location of the BTX Gemini Twin Waveform Electroporator should be a dry level sturdy surface free from extremes in ambient temperature dust or chemical exposures Unpack the safety dome cuvette rack and disposable cuvette chambers Connect the safety dome or in the case of the BTX Gemini X2 system specialty electrodes or HT plate handler into the connectors at the bottom right hand side of front panel Connect the mains power cord to into the back panel at the bottom left Power
48. le cells Biotechnol J October 4 10 1488 1496 2009 Rinaldi G Development of Functional Genomic Tools in Trematodes RNA Interference and Luciferase Reporter Gene Activity in Fasciola hepatica PLoS One 2 7 e260 July 2008 Nguyen K et al T Cell Costimulation via the Integrin VLA 4 Inhibits the Actin Dependent Centralization of Signaling Microclusters Containing the Adaptor SLP 76 Immunity 28 810 821 June 2008 Yang C et al Dimeric heat shock protein 40 binds radial spokes for generating coupled power strokes and recovery strokes of 9 2 flagella The Journal of Cell Biology 180 2 pp 403 415 January 28 2008 Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com Applications General References continued In Vitro Electroporation continued Zhao Y et al High Efficiency Transfection of Primary Human and Mouse T Lymphocytes Using RNA Electroporation Molecular Therapy Vo 13 No 1 January 2006 Heaney J D et al Tissue specific expression of a BAC transgene targeted to the Hprt locus in mouse embryonic stem cells Genomics 2004 Raoul C et al Motoneuron Death Triggered by a Specific Pathway Downstream of Fas Potentiation by ALS Linked SOD1 Mutations Neuron Vol 35 1067 1083 September 12 2002 Dawson K et al Insulin Regulated Trafficking of Dual Labeled Glucose Transporter 4 in Primary Rat Adipose Cells Biochemical
49. ll types while maintaining critical cell viability Transfection using this high performance electroporation solution is equally effective in delivering DNA as well as siRNA into mammalian cells PBS is a buffer solution commonly used in biological research It is a water based salt solution containing sodium chloride sodium phosphate and in some formulations potassium chloride and potassium phosphate The buffer s phosphate groups help to maintain a constant pH The osmolarity and ion concentrations of the solution usually match those of the human body isotonic HEPES is widely used in cell culture largely because it is better at maintaining physiological pH despite changes in carbon dioxide concentration produced by cellular respiration when compared to bicarbonate buffers which are also commonly used in cell culture RPMI is a form of medium used in cell culture and tissue culture It has traditionally been used for growth of Human lymphoid cells This medium contains a great deal of phosphate and is formulated for use in a 5 carbon dioxide atmosphere Opti MEM is an improved Minimal Essential Medium MEM that allows for a reduction of Fetal Bovine Serum supplementation by at least 50 with no change to growth rate or morphology Opti MEMt can be used with a variety of suspension and adherent mammalian cells including Sp2 AE 1 CHO BHK 21 HEK and primary fibroblasts MEM Eagle is suitable for a diver
50. load Generator to PC 1 Select Protocol s 2 Click Save Protocol P NOTE Hit Refresh to update the list after creating or editing protocols on the Gemini X generator 3 Protocol Manager Advanced Protocol Manager Installed Protocols Upload Protocols From Unit To PC Protocol Select Download Protocols From PC To Unit F COM Port VYas_smooth_musc COMS D COM Port List Refresh Protocol Send Unit Commands p ga Cancel Manage Log Files Pulse Data Refresh Delete Save Protocol 3 Select the drive and folder location A Click Save 5 EN v Ai Advanced Protocol Manager Save As Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com 29 HARVARD APPARATUS Protocol Manager Download PC to Generator 1 Click Choose Protocol NOTE If a protocol already exists on the Gemini X generator Protocol Manager will not overwrite the file if you attempt to 2 Select Protocol s to download re download it 3 Click Open otocol Manager Protocol Manager HI Adv d Protocol Manager Vv Ze Advanced Protocol Manager me x e E m wk Save in L Protocols D E Eup ET SQUARE BTX Installed Protocols Upload Protocols From Unit To PC 4 Protocol name being dowloaded is shown Y1 Protocol Manager Advanced Protocol Manager ARVARD AP
51. miting the generality of the foregoing HARVARD APPARATUS BTX shall not be liable for any claims of any kind whatsoever as to the equipment delivered or for non delivery of equipment and whether or not based on negligence Warranty is void if the BTX Gemini X2 amp BTX Gemini SC instrument is changed in any way from Its original factory design or if repairs are attempted without written authorization by HARVARD APPARATUS BTX Warranty is void if parts connections or electrodes not manufactured by HARVARD APPARATUS BTX are used with the BTX Gemini X2 amp BTX Gemini SC Instrument If a defect arises within the warranty period promptly contact Harvard Apparatus BTX 84 October Hill Road Building 7 Holliston Massachusetts USA 01746 1388 using our toll free number 1 800 272 2775 US Only or 508 893 8999 E mail techsupport btx harvardapparatus com Goods will not be accepted for return unless an RMA Returned Materials Authorization number has been issued by our customer service department The customer is responsible for shipping charges Please allow a reasonable period of time for completion of repairs replacement and return If the unit is replaced the replacement unit is covered only for the remainder of the original warranty period dating from the purchase of the original device This warranty gives you specific rights and you may also have other rights which vary from state to state Gemini Twin Waveform Electroportation Syst
52. multiple pulsing options with both the square waveform and the exponential decay waveform and unparalleled pulse delivery accuracy The BTX Gemini X with over 1 000 custom protocol storage monitors and displays pre pulse sample resistance as well as delivered voltage values and records logs of all experiment parameters internally which can be downloaded to a computer for analysis and QC The generator is controlled through a color LCD touch screen interface and incorporates USB communications The pulse can also be activated by a foot switch The enhanced safety features of the BTX Gemini X protect users as well as precious samples The BTX Gemini X2 is designed to give researchers the ultimate flexibility in their experiments making it possible to perform electroporation on tissues and organs in vivo as well as in utero in ovo ex plant on adherent cells and cells in suspension in either single cuvettes or 96 well plates The Safety Dome allows researchers to safely work with electroporation cuvettes up to two at a time giving researchers the ability to experiment on sample volumes from 20 ul up to 800 ul This state of the art system comes complete with the twin waveform generator for cell electroporation in all forms dozens of preprogramed protocols for commonly electroporated cells Safety Dome 30 cuvettes cuvette stand user manual two year warranty unlimited application support and the same high quality researchers have come t
53. ncorporation Verify physical biological chemical parameters Verify delivery of the pulse and pulse parameters Is the voltage correct Chamber gap Pulse length or appropriate instrument settings Number of pulses If so follow Optimization Guidelines outlined Low Viability Verity physical biological chemical parameters Is the voltage chamber gap pulse length time constant pulse number and other instrument settings correct If so reduce voltage pulse length or number of pulses and re optimize protocol to improve viability as outlined Voltage Drop A drop in output voltage accompanies pulse delivery into highly conductive samples for example PBS Thus the displayed voltage may in these situations be less than that expected given 5 full scale accuracy and the monitoring accuracy of 5 Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com 43 HARVARD APPARATUS Ordering Information Order No Description Order No Description Electroporation Systems Accessories 45 2001 BTX Gemini SC Generator only ea 45 0400 HT 100 Plate Handler Manual ea 45 2002 BTX Gemini SC Electroporation System ea 45 0401 HT 200 Plate Handler Auto ea Includes Gemini SC Generator Cuvett
54. nued Preset Protocols Square Wave continued On the home screen tap the Preset Protocols icon View your protocol HARVARD APPARATUS 02 26 13 9 51 28 AM To run tap the Omega icon Preset Protocols RUN PROTOCOL HARVARD APPARATUS M _NEW_PROTOCOL Square Wave Set Meas 04 02 13 4 06 40 PM Voltage v 200 Duration ms 1 Resistance ohm Droop Plate of Pulses 2 Handler Pulse Interval s 1 0 Gap width mm 4 Meas Load ohm Tha Double tap desired cell type User Protocols ZPS t HARVARD APPARATUS 02 26 13 1 22 11 PM RUN PROTOCOL HARVARD APPARATUS M _NEW_PROTOCOL SSS EE Square Wave Meas 04 02 13 4 06 58 PM Voltage v Resistance ohm Droop 0 0 Plate of Pulses 2 Handler gt o Pulse Interval s 1 0 Gap width mm 4 Meas Load ohm OK d lt TH O 4 Scroll through available cells Double tap desired cells Once protocol is complete data regarding your pulse is displayed HARVARD APPARATUS and stored in the systems logs BTX Gemini X only for future use Mammalian 02 26 13 1 24 37 PM You may continue pulsing go back to the set up screen or to the home screen RUN PROTOCOL HARVARD APPARATUS big NEW DROTOCUL Kam Square Wave Meas 04 02 13 4 07 39 PM Voltage v 206 Pulses delivered 2 Duration ms 1 Resistance ohm Droop 00 00 Plate PRESS TO CLEAR MESSAGE of Pulses 2 Handler Pulse Interval s 1 0 Gap width mm 4 Meas Load ohm
55. o expect from Harvard Apparatus BIX HARVARD APPARATUS BTX The BTX Gemini SC is a twin waveform generator incorporating both square and exponential decay waves in a single unit These waveform combinations enable researchers to easily and efficiently electroporate eukaryotic and prokaryotic cells in suspension with one easy to use setup The BTX Gemini SC boasts a wide range of voltage 10 3000 v 5 v or 10 v increments pulse length 50 uS 5 mS 50 US resolution time constant options which include capacitance choices from 25 to 3275 uF in LV and 10 25 or 50 uF in HV multiple pulsing option with the square waveform and unparalleled pulse delivery accuracy The BTX Gemini SC with unlimited custom protocol storage monitors and displays pre pulse sample resistance as well as delivered voltage values The generator is controlled through a color LCD touch screen interface The enhanced safety features of the BTX Gemini SC protect users as well as precious samples The Safety Dome allows researchers to safely work with electroporation cuvettes up to two at a time giving researchers the ability to experiment on sample volumes from 20 ul up to 800 ul This affordable system comes complete with the dual waveform generator for suspension cell electroporation dozens of preprogramed protocols for commonly electroporated cells Safety Dome 30 cuvettes cuvette stand user manual two year warranty unlimited application support and the s
56. of Hepatocellular Carcinoma Cancer Research 70 4 February 15 2010 Roos A K Skin Electroporation Effects on Transgene Expression DNA Persistence and Local Tissue Environment PLoS ONE 4 9 e7226 2009 Brave A et al Late administration of plasmid DNA by intradermal electroporation efficiently boosts DNA primed T and B cell responses to carcinoembryonic antigen Vaccine 27 3692 3696 2009 Roos A K et al Optimization of Skin Electroporation in Mice to Increase Tolerability of DNA Vaccine Delivery to Patients Molecular Therapy 17 9 1637 1642 Sep 2009 Danner S et al Seminiferous tubule transfection in vitro to define post meiotic gene regulation Reproductive Biology and Endocrinology 7 67 2009 HARVARD APPARATUS BTX Benton C et al Modest PGC 1_ Overexpression in Muscle in Vivo Is Sufficient to Increase Insulin Sensitivity and Palmitate Oxidation in Subsarcolemmal Not Intermyofibrillar Mitochondria The Journal of Biological Chemistry 283 7 pp 4228 4240 February 15 2008 Chesler A T Selective Gene Expression by Postnatal Electroporation during Olfactory Interneuron Neurogenesis PLoS ONE 3 1 e1517 2008 Rao N M et al Electroporation of Adult Zebrafish S Li ed Electroporation Protocols Preclinical and Clinical Gene Medicine Methods in Molecular Biology Vol 423 p 289 2008 Johnson C J et al Technical Brief Subretinal injection and electroporation into adult mou
57. or driver software on your computer Search for driver software in this location Deevers J Include subfolders gt Let me pick from a list of device drivers on my computer This list will show installed driver software compatible with the device and all driver software in the same category as the device 5 If a warning message regarding the driver publisher is displayed choose Install this driver software anyway zb Don t install this driver software You should check your manufacturer s website for updated driver software T gt Install this driver software anyway Only install driver software obtained from your manufacturer s website or disc Unsigned software from other sources may harm your computer cr steal information v See details Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com 34 Up grading BIX Gemini Series Software Device Updater Program ZA 1 NOTE Prior to upgrading users will need to install USD drivers as well as the bootloader driver file Reference Virtual Comport Driver Installation Upload the latest software versions to your desktop format is filename srec There are two srec files that both need to be updated BTX Gemini vx x x and a BTX Gemini Pwr VX X X Disconnect all I O devices and then connect the Gemini X2 generator to PC using a USB cable On the Set u
58. p Bank C Max Charge capacitor bank C voltage is above tolerance Turn off the unit for a few minutes then turn back on Re run the protocol if the issue recurs contact the manufacturer Charge Cap Bank C Min Charge capacitor bank C voltage is below tolerance Turn off the unit for a few minutes then turn back on Re run the protocol if the issue recurs contact the manufacturer Dump Time Max The capacitor bank did not fully discharge in the estimated time Turn off the unit for a few minutes then turn back on Re run the protocol if the issue recurs contact the manufacturer Invalid Protocol System Processing Busy The generator was requested to run an invalid protocol The control command is invalid or was issued while the generator was busy Verify that the used protocol is within the allowed specifications of the generator Allow more time between events TC_Error The measured rc time while delivering an exponential decay pulse is invalid Hardware error Please contact the manufacturer Pulse Duration Error The pulse duration did not meet the expected duration Hardware error Please contact the manufacturer Unit Type Sel Error The unit hardware and software do not match the corect type Hardware error Please contact the manufacturer Error OX Multiple errors Multiple error Please contact the manufacturer Gemini Twin Waveform Elec
59. p screen of the Gemini X generator press the Upgrade Software button twice to enter Boot Loader Mode ya NOTE To exit boot loader mode without upgrading the software power cycle the generator From the CD provided with the Gemini X generator open the Device Updater Application Za Device Updater device is connected in Update Software mode Open Update File GAUpdate Files BTX Gemini 2 0 0 sree 89100900313120456C6974652E1737265639F Click Open Update File and browse to the BTX Gemini vx x x srec file that was previously saved to the desktop Click Start Update Za Device Updater device is connected in Update Software mode G Update Files BTX Gemini v2 0 0 srec S01L0000031 512 0456C 6574652573726563 9P Downloaded file 1319006 bytes Complete comMm3 _ After the update is complete press Reboot and then power cycle the Gemini X generator from the power switch in the back of the unit Repeat steps 5 through 7 with the BTX Gemini Pwr VX X X Srec Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com 35 HARVARD APPARATUS BTX Troubleshooting NOTE It is recommended that you save your protocols BZA periodically by uploading them to a PC Out of Range A value was entered or encountered in a protocol that was beyond the generator s limits SET PROTOCOL VOLTAGE HARVA
60. pa Ke Se Sint S MUN Lee 45 0106 Microslide 453 10 10 mm gap 2 0 ml Ea GE GE Ki 45 0107 Meander Fusion Chamber See gap pk 4 4 Pk E E i 45 0502 Tissue Slice Wand 10 mm Ea 45 0108 Ce Field mm Ea needs 45 0501 45 0503 45 0204 45 0217 Electrode Cable for Flat Electrode 10 ft BTB Ea bains GE Reeg SES 43 0107 Fatpack Chambers 1 83 mm gap pk 50 SOTK 45 0530 Adherent Cell Electrode 3 mm gap Ea 45 0110 Flatpack Chambers 0 56 mm gap pk 50 50 Pk needs 45 0204 45 0113 Genetrodes Straight 5 mm Gold Tip Ea 45 0531 Adherent Cell Electrode 3 mm Kit Ea 45 0160 Genetrodes Straight 5 mm Gold Tip Kit Ea 45 0486 PT Tweezertrodes mm Diameter Kit Ea 45 0114 Genetrodes Straight 10 mm Gold Tip Ea 45 0487 PT Tweezertrodes 3 mm Diameter Kit Ea 45 0161 Genetrodes Straight 10 mm Gold Tip Kit Ea 45 0489 PT Tweezertrodes 5 mm Diameter Kit Ea 45 0115 Genetrodes L Shape 5 mm Gold Tip Ea 45 0488 PT Tweezertrodes 7 mm Diameter Kit Ea 45 0162 Genetrodes L Shape 5 mm Gold Tip Kit Ea 45 0165 SS Tweezertrode 7 mm Diameter Kit Ea 45 0116 Genetrodes L Shape 3 mm Gold Tip Ea 45 0118 SS Tweezertrode 7 mm Diameter needs 45 0204 Ea 45 0163 Genetrodes L Shape 3 mm Gold Tip Kit Ea 45 0166 SS Tweezertrode 10 mm Diameter Kit Ea 45 0117 Genetrodes L Shape mm Gold Tip Ea 45 0119 SS Tweezertrode TO mm Diameter Ea 45 0164 Genetrodes L Shape mm Gold Tip Kit Ea needs 45 0204 45 0203 Gene
61. peglab a VWR company Gemini Twin Waveform Electroportation systems Users s Manual HARVARD APPARATUS BTX www btxonline com 5507 002 Rev A RESEARCH ONLY Harvard Apparatus BTX 84 October Hill Rd Holliston MA 01746 USA Phone 1 508 893 8999 Fax 1 800 429 5732 Web www btxonline com Warranty Harvard Apparatus warranties the BTX Gemini X2 amp BTX Gemini SC Twin Waveform Electroporation Systems for a period of two years from the date of purchase At its option Harvard Apparatus BTX will repair or replace the unit if it is found to be defective as to workmanship or materials This warranty does not extend to any instrumentation which has been a subjected to misuse neglect accident or abuse b repaired or altered by anyone other than HARVARD APPARATUS BTX without HARVARD APPARATUS BTX express and prior approval c used in violation of instructions furnished by HARVARD APPARATUS BTX This warranty extends only to the original customer purchaser IN NO EVENT SHALL HARVARD APPARATUS BTX BE LIABLE FOR INCIDENTAL OR CONSEQUENTIAL DAMAGES Some states do not allow exclusion or limitation of incidental or consequential damages so the above limitation or exclusion may not apply to you THERE ARE NO IMPLIED WARRANTIES OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR USE OR OF ANY OTHER NATURE Some states do not allow this limitation on an implied warranty so the above limitation may not apply to you Without li
62. ponential decay wave system is called the time constant which is characterized by the rate at which the pulsed energy e or voltage is decayed to 1 3 the original set voltage This time constant is modified by adjusting the resistance and capacitance RC values in an exponential decay wave form Time constant calculation T RC where T is time and R is resistance and C is capacitance The pulse length works indirectly with the field strength to increase pore formation and therefore the uptake of target molecules Generally during optimization of parameters an increase in voltage should be followed by an incremental decrease in pulse length When decreasing the voltage the reverse Is true Pulse length is a key variable that works hand in hand with voltage and needs to be considered when optimizing electrical parameters to maximize the results for a given cell type Number of Pulses Electroporation is typically carried out as a single pulse for most cell types However other cell lines may require multiple pulses to achieve maximum transfection efficiencies Usually lower voltages are used when applying multiple pulses in order to gradually permeate the cell membranes This allows the transfer of molecules while avoiding damage to delicate or whole tissue samples This method of multiple pulsing is critical for maximum gene delivery without causing tissue damage to Im vivo in utero and explant tissue environments The use of multiple
63. pulse will require the optimization of key electrical parameters including voltage and pulse length Typically for in vivo applications the use of lower voltages between 10 100 volts with pulse lengths ranging 30 50msec provides efficient transfection The optimal voltage pulse length and number of pulses will vary depending on the cell type and molecule DNA or RNA transfected Electroporation Buffer The buffers used for electroporation can vary depending on the cell type Many applications use highly conductive buffers such as PBS Phosphate Buffered Saline lt 30 ohms and HBSS Hepes Buffer lt 30 ohms or standard culture media which may contain serum Other recommended buffers are hypoosmolar buffers in which cells absorbs water shortly before pulse This swelling of the cells results in lowering the optimal permeation voltage while ensuring the membrane is more easily permeable for many cells but can be damaging to others Prokaryotic cells such as bacteria require the use of high resistance buffers gt 3000 ohms for this reason proper preparation and washing of the cells is essential to remove excess salt ions to reduce the chance of arcing lonic strength of an electroporation buffer has a direct affect on the General Optimization Guide for Electroporation HARVARD APPARATUS BTX resistance of the sample which in turn will affect the pulse length or time constant of the pulse The volume of liquid in a cuvette has significan
64. rial to treat external tumors Research has shown that the induction of pathways Is affected by three major factors First cell to cell biological variability causes some cells to be more sensitive to electroporation than other cells Second for pathways to be induced the product of the pulse amplitude and the pulse duration has to be above a lower limit threshold Third the number of pathways and effective pathway diameter increases with the product of amplitude and duration Although other factors are involved this threshold is now understood to be largely dependent on a fourth factor the reciprocal of cell size If the upper limit threshold is reached pore diameter and total pore area are too large for the cell to repair by any spontaneous or biological process the result is irreversible damage to the cell or cell lysis Because the mechanism of electroporation is not well understood the development of protocols for a particular application has usually been achieved empirically by adjusting pulse parameters amplitude duration number and inter pulse interval Research shows that certain experimental conditions and parameters of electrical pulses may be capable of causing many more molecules to move per unit time than simple diffusion There is also good evidence Sukharev et al 1992 that DNA movement is in the opposite direction An additional important consideration is when the voltage pulse is applied to the cell
65. rotocol at any time P NOTE Generator will display readings of the 1st and last pulse of each column RUN PROTOCOL HARVARD APPARATUS Rat embryo 563 SE Square Wave Set Meas 04 03 13 10 44 22 AM Voltage v 33 Delivering Pulse Duration ms 50 E PRESS TO CLEAR MESSAGE Resistance ohm Droop 1 3 of Pulses 5 Pulse Interval s 1 0 Gap width mm 4 Meas Load ohm OK y PI e lt P gt t Once protocol is complete data regarding your pulse is displayed and stored in the systems logs for future use You may continue pulsing go back to the settings screen or protocol select screen or to the home screen RUN PROTOCOL HARVARD APPARATUS Rat embryo 563 SE Square Wave Set Meas 04 03 13 10 44 54 AM Voltage v 35 32 2 Pulses delivered 5 Duration ms 50 50 Resistance ohm Droop 13 E Plate of Pulses 5 Handler Pulse Interval s 1 0 Gap width mm 4 Meas Load ohm OK tri DEI PRESS TO CLEAR MESSAGE Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com 22 Performing Experiments Using Specialty Electrodes BTX Gemini X only Plug the specialty electrodes into the front ports of the BTX Gemini X2 Scroll through available protocols Double tap desired selection HARVARD APPARATUS 02 27 13 9 43 36 AM 600 Scroll through available cells Double tap desired cells HARVARD APPARATUS 02 27 13 9 45 20 AM
66. s and medium that the amount of current that flows is dependent on the conductivity of the material in which the cells are located Some material is quite conductive and severe heating will occur if the pulse duration is too long Therefore long duration fields will kill cells by destroying the membrane and heating HARVARD APPARATUS BTX The electric field in which the cells are located is produced by two system components The first is the voltage waveform generator and the second is the electrode which converts the voltage into the electric field As the charge accumulates at the membrane which is a capacitance the voltage across the membrane increases voltage capacitance charge As charge accumulates at the membrane the voltage across the membrane Increases Neumann et al 1989 described the equation that relates the transmembrane voltage TMV to electric field intensity TMV 32 E r cos al where E electric field intensity in volts cm r the cell radius in cm a angle off the center line Pores in the membrane will begin to form as the voltage increases from its quiescent value of a few tenths of a volt to more than 0 5 volts To produce a TMV of 1 volt across the membrane of a cell with 7um radius the required electric field intensity is 1 2 E k k 3 7x10 4 950 volts cm The number of pores and effective pore diameter increase as the product of pulse amplitude and duration increase
67. se eyes Molecular Vision 14 2211 2226 2008 Heller L et al Comparison of electrically mediated and liposome complexedplasmid DNA delivery to the skin Genetic Vaccines and Therapy 6 16 2008 Roos A K et al Enhancement of Cellular Inmune Response to a Prostate Cancer DNA Vaccine by Intradermal Electroporation Molecular Therapy 13 2 February 2006 Kong X C et al Inhibition of synapse assembly in mammalian muscle in vivo by RNA interference EMBO Rep 5 2 183 188 January 2004 Pringle I A et al Duration of reporter gene expression from naled pDNA in the mouse lung following direct electroporation and development or wire electrodes for sheep lung electroporation studies Molecular Therapy 9 S56 S56 2004 Mikata K et al Inhibition of Growth of Human Prostate Cancer Xenograft by Transfection of p53 Gene Gene Transfer by Electroporation Molecular Cancer Therapeutics Vol 1 247 252 February 2002 Pekarik V et al Screening for gene function in chicken embryo using RNAi and electroporation Nature Biotechnology 21 93 96 December 2002 Dujardin N et al In vivo assessment of skin electroporation using square wave pulses J Controlled Release 79 219 227 2002 Drabick J J et al Cutaneous Transfection and Immune Responses to Intradermal Nucleic Acid Vaccination Are Significantly Enhanced by in Vivo Electropermeabilization Molecular Therapy 3 2 Feb 2001 In Utero Electroporation M
68. se spectrum of mammalian cell types Various formulations available with either Hank s or Earle s salts DMEM MEM is used in a wide range of mammalian cell culture applications The high glucose version is well suited to high density suspension culture The low glucose formula is used for adherent dependent cells CytoMix is a composition of cytokines for the highly efficient and reproducible expansion of human multipotent mesenchymal stromal cells MSCs HARVARD APPARATUS BTX Water amp 10 glycerol Typically used for bacteria Cytoporation Media T is a buffer designed for larger volume cell electroporation as it incorporates a low conductivity of 0 08 S cm to reduce heating of solution during electroporation Cytoporation Media T4 is a buffer designed for larger volume cell electroporation as it incorporates a low conductivity of 3 45 mS cm to reduce heating of solution during electroporation Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com 42 Experiment Troubleshooting Arcing Verify electrical component functionality Verify properties of cell sample do cells need to be washed Is the buffer appropriate for application Verify properties of transfectant molecule Is the DNA well purified Try reducing the voltage or increase sample volume until arcing is no longer a problem Low or no Transfection Efficiency or I
69. size 2 Reducing the volume 3 Replace the buffer with less conductive type of buffer Over Current Pulse Abort Pulse aborted due to pulse over current The over current protection feature is added to prevent sample arcing This could occur when the sample resistance changes during the pulse Sample resistance is very low for the voltage selected 2 Gap size is too small for the voltage selected 3 Pulse duration is too long Idle V Max Voltage or current in idle state is higher than expected Turn off the unit for a few minutes then turn back on Re run the protocol if the issue recurs contact the manufacturer Charge Cap Bank A Max Charge capacitor bank A voltage is above tolerance Turn off the unit for a few minutes then turn back on Re run the protocol if the issue recurs contact the manufacturer Charge Cap Bank A Min Charge capacitor bank A voltage is below tolerance Turn off the unit for a few minutes then turn back on Re run the protocol if the issue recurs contact the manufacturer Charge Cap Bank B Max Charge capacitor bank B voltage is above tolerance Turn off the unit for a few minutes then turn back on Re run the protocol if the issue recurs contact the manufacturer Charge Cap Bank B Min Charge capacitor bank B voltage is below tolerance Turn off the unit for a few minutes then turn back on Re run the protocol if the issue recurs contact the manufacturer Charge Ca
70. sssasssssssssssac saes sazscassesesscgsasgs ee ANE SE aa A E 43 Ordering T T E aT 44 45 Declaration Of Conformity esse sees sees eee eee ee eee 46 Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com 3 Safety Information Please read the following safety precautions to ensure proper use of your generator If the equipment is used in a manner not specified the protection provided by the equipment may be impaired To Prevent Hazard or Injury Use Proper Line Cord Use only the specified line cord for this product and make sure line cord is certified for country of use The operating voltage range for the BTX Gemini Twin Wave Series is 100 240 vac 50 60 Hz Ground the Product This product is grounded through the grounding conductor of the power cord To avoid electric shock the grounding conductor must be connected to earth ground Before making any connections to the input or output terminals of the product ensure that the product is properly grounded Make Proper Connections Make sure all connections are made properly and securely Any signal wire connections to the unit must be no longer than 3 meters Observe All Terminal Ratings Review the operating manual to learn the ratings on all connections Use Proper Fuse Use only specified fuses with product Avoid Exposed Circuitry Do not touch any electronic circuitry inside of the product Do Not
71. t 45 02 6 45 0217 pr 45 0466 25 Well Plate 2 mm gap 125 U ea 45 0090 Adapter Set Banana to Pin Tip ea 45 0467 25 Well Plate 2 mm gap 12 gt Ul pk 6 6 pk 45 0089 Banana to Square Post Adapter Set for 45 02 17 pr 45 0450 96 Well Plate 2 mm gap 125 ul ea 45 0452 96 Well Plate 4 mm gap 250 ul ea Reagents 45 0802 BTXpress 5 ml ea 45 0803 BTXpress 5 ml with 2 mm gap Cuvettes pk 50 ea 45 0804 BTXpress 5 ml with 4 mm gap Cuvettes pk 20 ea 45 0805 BTXpress 10 ml ea 45 0806 BTXpress 10 ml with 2 mm gap Cuvettes pk 50 ea 45 0807 BTXpress 10 ml with 4 mm gap Cuvettes pk 40 ea 47 0002 Cytoporation Media T 500 ml ea 47 0003 Cytoporation Media T4 500 ml ea Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com 44 HARVARD APPARATUS BTX Ordering Information Order No Description UOM Order No Description UOM 45 0101 Caliper Electrode 1 0 x 1 0 cm Kit Ea 45 0491 Tissue Slice Chamber 7 x 7 mm Dish Ea 45 0102 Caliper Electrode 2 0 x 2 0 cm 1 5x 5cmKit I Ea needs 45 0492 45 0503 45 0204 45 0103 Microslide 450 0 5 mm gap 20 ul pk 10 10 Pk 45 0492 Tissue Slice Wand 7 mm Ea 45 0104 Microslide 450 1 mm gap 40 ul pk 10 10 Pk E 45 0105 Microslide 453 3 2 mm gap 650 ul Ea
72. t effect on sample resistance for ionic solutions the resistance of the sample is inversely proportional to the volume of solution and pH As the volumes are increased resistance decreases which increases the chance of arcing while lowering the volume will increase the resistance and decrease the arc potential Harvard Apparatus BTX now offers BTXpress High Performance Electroporation Solution a low conductance buffer that achieves higher transfection efficiencies with minimal cell toxicity The BTXpress buffer is a single buffer developed to facilitate high efficiency gene delivery into mammalian cells DNA RNA Concentrations Electroporation is typically thought of as a nucleic acid DNA mRNA siRNA and miRNA transfer method into prokaryotic and eukaryotic cells Electroporation is not limited to just nucleic acid delivery it can introduce proteins antibodies small molecules and fluorescent dyes The standard range of DNA used for transfections is 5 20g ml for most cell types however in some instances increasing the DNA concentration as high as 50g ml improves transfection efficiency without changing other parameters Determining the optimal DNA concentration through a DNA titration can be beneficial The size of a molecule will have an effect on the electrical parameters used to transfect the cell Smaller molecules siRNA or miRNA may need higher voltage with microsecond pulse lengths and larger molecules DNA may need lower voltag
73. tance ohm 200 Capacitance uF 25 Plate of Pulses 1 Handler gt o Pulse Interval s Gap width mm 2 Meas Load ohm OK K o TC t Once protocol is complete data regarding your pulse is displayed and stored in the systems logs BTX Gemini X only for future use You may continue pulsing go back to set up screen or to the home screen MY_NEW_ PROTOCOL Gap HARVARD APPARA E DO LS tial 2 HARVARD APPARATUS BZ NOTE See the Protocol Manager user manual for instructions on viewing and downloading log data RUN PROTOCOL HARVARD APPARATUS MY_NEW_PROTOCOL Se ei RE Exponential Decay Set Meas 04 02 13 3 32 58 PM Voltage v 3000 2940 Pulses delivered 1 Time Const ms 4 18 44 Resistance ohm 200 Capacitance uF 25 Plate of Pulses 1 Handler Pulse Interval s Gap width mm 2 Meas Load ohm OK 1012 OO PRESS TO CLEAR MESSAGE Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com 16 HARVARD APPARATUS BTX Performing Experiments Use the numeric keypad to set voltage New Protocols continued Square Wave Tap the Green Check icon to save Tap the Square Wave icon SET PROTOCOL VOLTAGE F nm Tap the Green Check icon to save Voltage 02 26 13 4 22 59 PM 29o OO En PROTOCOL GAP Co HARVARD APPARATUS ae ave a 02 26 13 4 24 55 PM HARVARD APPARATUS 02 26 13 4 19 43 PM 4 19 43 PM e HEK
74. timicrobial Agents Chemotherapy 50 4 1342 1346 April 2006 Bindu Garg Romesh C Dogra and Parveen K Sharma High Efficiency Transformation ofRhizobium leguminosarum by Electroporation Applied Environmental Microbiology 65 6 2802 2804 June 1999 General References In vitro Electroporation Kim T et al Mesoporous Silica Coated Hollow Manganese Oxide Nanoparticles as Positive T1 Contrast Agents for Labeling and MRI Tracking of Adipose Derived Mesenchymal Stem Cells J Am Chem Soc 133 2955 2961 2011 Kataoka N et al Development of butanol tolerant Bacillus subtilis strain GRSW2 B7 as a potential bioproduction AMB Express 1 10 2011 Hutson T H et al Optimization of a 96 well electroporation assay for post natal rat CNS neurons suitable for cost effective medium throughput screening of genes that promote neurite outgrowth Frontiers in Molecular Neuroscience 4 55 December 2011 Djouad F et al Activin A expression regulates multipotency of mesenchymal progenitor cells Stem Cell Res amp Therapy 1 11 2010 Sankaranarayanan K et al Electro Molecular Therapy using Adult Mesenchymal Stem Cells Proc ESA Annual Meeting on Electrostatics 13 2010 Blackmore M et al High content screening of cortical neurons identifies novel regulators of axon growth Molecular and Cellular Neuroscience 44 43 54 2010 Yao S et al Improvement of electroporation to deliver plasmid DNA into dental follic
75. to decrease to a value 1 e of the true peak pulse Transfection The introduction of nucleic acids into animal cells Stable transfections result in integration of nucleic acids into host chromosomes and the inheritance of associated traits in progeny cells Transient transfections result in temporary expression of exogenous nucleic acids Transformation The introduction of nucleic acids into microorganisms and plant cells Turgor Pressure The pressure in capillaries Voltage The difference of electric potential between two electrodes expressed in volts V or kilovolts kV Waveforms The shape of time varying electric signals Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com Product Overview The BTX Gemini X2 is a highly advanced twin waveform generator Incorporating both square and exponential decay waves in a single unit The BTX Gemini X2 has been designed with these waveform combinations to enable researchers to easily and efficiently electroporate eukaryotic cells and prokaryotic cells in all forms with one easy to use setup The BTX Gemini X2 which can be operated via PC or remote control boasts a wide range of voltage 5 3000 v 1 v or 5 v increments pulse length 10 uS 1S 1 uS resolution time constant options which include capacitance choices from 25 to 3275 uF in LV and 10 25 35 50 60 75 85 uF in HV along with
76. trodes Genepaddle Holder with Shaft Ea 45 0524 PT Tweezertrode mm Flat needs 45 0204 Ea 45 0216 Genetrodes Genepaddle Cable 10 ft BTMG Ea 45 0525 PT Tweezertrode mm Flat Kit Ea 45 0122 Genepaddles 3 x 5 mm Ea 45 0204 Tweezertrode Cable Single Adaptor Cable Ea 45 0169 Genepaddle 3 x 5 mm Kit Es for Tissue Slice Electrode Positive 45 0170 Genepaddle 5 x 7 mm Kit Ea 47 0090 Electrode Adapter Box for Agile Pulse In Vivo Ea 45 0167 2 Needle Array 10 mm Kit Ea 47 0000 Parallel Needle Array Handle for AP In Vivo Ea 45 0205 2 Needle Array Handle 10 mm needs 45 0120 Ea 47 0040 4 Needle Array 4 mm gap 2 mm Length Ea 45 0120 2 Needle Array 10 mm pk 6 needs 45 0205 Ea AP In Vivo ID 45 0168 2 Needle Array 5 mm Kit Ea 47 0043 3 Needle Array 4 mm gap 3 mm Length Ea 45 0206 2 Needle Array Handle 5mm needs 45 0121 1 Ea aoe 45 0121 2 Needle Array 5 mm pk 6 needs 45 0206 Ea oes ees S MZ 2 K 45 0510 Needle L Shaped Pt Electrode 3 mm Kit Ea 47 0050 PIN Gece sia EE E 45 0509 Needle L Shaped Pt Electrode 3 mm Ea AP In Vivo ID Ua EE 47 0060 6 Needle Array 6 mm gap 2 mm Length Ea 45 0513 Petri 7 mm Tissue Chamber Kit Ea AP In Vivo ID 45 0505 Petri Dish Tissue Chamber 5 x 5 mm Kit Ea 47 0070 6 Needle Array 6 mm gap 10 mm Length Es 45 0504 Petri Dish Tissue Chamber 5 x 5 mm Ea AP In Vivo IM needs 45 0216 47 0080 6 Needle Array 6 mm gap 12 mm Length Ea 45 0506
77. troportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com 2 All Preset and User Defined Protocols are stored in the Protocol List for ongoing use Over time you may want to manage previously created Protocols to keep your Protocol List easy to navigate In addition the BTX Gemini X2 allows you to export Protocol information to an externally connected generator or to a computer that will be used to control operation This section of the Guide describes the file management facilities available on the BTX Gemini X2 PROTOCOL SELECT Bacteria Yeast 15 Protocols HARVARD APPARATUS 06 06 13 16 25 54 D pastoris E C3 gw DER Saving a Copy of a Protocol Follow these instructions to save a copy of a Protocol under a new name S cerevisiae C oleophila P aeruginosa C synechocystis 1 From Protocol Configuration screen choose the Protocol Name button to enter the Mode Selection screen 2 From the list of available Protocols select press once the Protocol you want to save a copy of then choose the File Options button to enter the File Options screen 3 Choose Save As Copy to access the Protocol Name Entry screen The current Protocol Name will be displayed in the text entry box 4 On the Protocol Name screen type the new name for your Protocol using the onscreen keyboard Choose the Symbols Numbers button 123 to display the list of avail
78. us BTX technicians Repair Facilities and Parts Harvard Apparatus BTX stocks replacement and repair parts When ordering please describe parts as completely as possible preferably using our part numbers If practical enclose a sample photo or drawing Caution Notice The BTX Gemini X2 amp BTX Gemini SC systems are intended for laboratory use only and can be used In research and development applications These systems have been designed to meet the standards for electromagnetic compatibility EMC intended for laboratory equipment applications The BTX Gemini X2 amp BTX Gemini SC systems comply with the applicable sections of IEC 61010 1 2012 3rd Ed Safety requirements for electrical equipment for measurement control and laboratory use This product should not be used in the presence of a flammable atmosphere such as an anesthetic mixture with air oxygen or nitrous oxide Gemini Twin Waveform Electroportation System User s Manual toll free 800 272 2775 s 508 893 8999 e www btxonline com HARVARD APPARATUS BTX Table of Contents SUBJECT PAGE Salei T Ten E 4 Generat r ZEIEN Ee EE 5 6 Tg sie Ee TT ete E ee E E E E ee 7 Eleciroporation Ehe aaea ee 8 Gossa OE SUNS eer 9 Product OVS E 10 Unpacking the System ccs ees eee eee eee eee eee 11 Op rating the E arusasa Seca eda cans canned neansneunendidavideiendensndenandenavecemndenns 12 13 UNM MM EE 12 Setting Audible Alarm Preterences cccccccccccccccee
79. variables Other parameters which play a crucial role in optimization include cell diameter plasmid concentrations temperature and electroporation buffer Wave Forms Pulse shape generally falls into two categories square wave or exponential decay wave Square Wave Pulse Square wave pulses rise quickly to a set voltage level maintains this level during the duration of the set pulse length and quickly turns off Square waves yields higher efficiencies and viabilities in mammalian cells Square wave EP in in vivo and ex vivo tissues embryo s and plant protoplast applications yield better results in comparison to an exponential decay wave Exponential Decay Wave Pulse Exponential decay waves generate an electrical pulse by allowing a capacitor to completely discharge A pulse is discharged into a sample the voltage rises rapidly to the peak voltage set then declines over time The powerful exponential decay wave pulse is routinely used for transformation of gram negative and gram positive bacterial yeast plant tissues insect cells and some mammalian cells Field Strength The field strength is measured as the voltage delivered across an electrode gap and Is expressed as kV cm Field strength is critical to surpassing the electrical potential of the cell membrane to allow the temporary reversible permeation or pore formation to occur in the cell membrane Three factors should be considered for optimizing field strength

Gemini Twin Waveform Electroportation Systems Users`s Manual

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