Bio-rad Gene Pulser Xcell™ Electroporation Systems User Manual

Gene Pulser Xcell™Electroporation SystemInstruction ManualCatalog Numbers165-2660, 165-2661, 165-2662, 165-2666,165-2667, and 165-2668For technical se

2.2 Setting Up the System2.2.1 Setting Up the Gene Pulser Xcell Main Unit and Connecting the ShockPod (Cat. #s 165-2660, 165-2661, 165-2662, and 165-2

Fig. 2.2. Connecting the ShockPod to the Gene Pulser Xcell main unit. 2.2.2 Connecting the PC Module to the Gene Pulser Xcell Main Unit (Cat. #s 165

2.2.3 Connecting the CE Module to the Gene Pulser Xcell Unit (Cat. #s 165-2660, 165-2661, and 165-2667)The PC Module and CE Module may be connected to

Fig. 2.5. ShockPod with cuvette.The plastic panel covering the electrode may be removed for cleaning should the electrode becomedirty. To clean the u

Section 3Gene Pulser Xcell™Operating Instructions3.1 Section OverviewThis section describes the operation of the Gene Pulser Xcell. The following sum

3.2 Front Panel and Home Screen3.2.1 Description of the keypadSee Figure 3.1 for a view of the Gene Pulser Xcell front panel.Alpha-numeric keys This

Fig. 3-1. Gene Pulser Xcell front panel. See Section 3.2.1 for an explanation of the key functions.3.2.2 Home ScreenUpon turning on the power to Gen

8. Data management: allows the user to access pulse parameters and results for thelast 100 pulses logged by date and time9. Measurements: allows the

3.3 Manual Operation3.3.1 Manual Operation (Quick Guide)• From the Home screen:• Press Enter to select exponential decay;• Press 2, then Enter to sel

When the Resistance value indicates infinity, no resistors from the PC Module are used. The Measurementsfunction of Gene Pulser Xcell may be used to d

WarrantyThe Gene Pulser Xcell electroporation system is warranted against defects in materials and workmanshipfor 1 year. If any defects occur in the

• With the Protocol Detail screen on the LCD display another pulse can be delivered using the samepulse parameters. To change the pulse conditions, pr

Time Constant Protocol Detail screenFig. 3.4. Time Constant Protocol Detail Screen. This screen shows the parameters that may be specified for expone

• The following combination of parameters may be entered:Pulse length + VoltagePulse length + Voltage + Number of pulses + Pulse intervalSquare Wave P

• When the necessary parameter values have been specified, a flashing “P” appears in the characterspace in the lower right corner of the LCD display i

18Exponential Decay: Results ScreenFig. 3.6. Exponential Decay Protocol Results screen. The graph shows the exponential decaypulse. The table gives

3.3.6 Saving a program from Manual OperationA protocol created in manual mode may be saved as a User Protocol as follows. • Create a protocol as des

User Protocols screenFig. 3.10. User Protocols screen. This is an example of the first User Protocols screen. Togglebetween this screen and the secon

Warning screen: overwrite protocolFig. 3.11. Warning screen: overwrite protocol.• The default selection is “No”. Press Enter if you do not want to ov

3.4.2 Electroporation using Pre-set ProtocolsThere are nine Pre-set Bacterial Protocols, six Pre-set Fungal Protocols, and 12 Pre-set MammalianProtoco

Pre-Set Protocols ScreenFig. 3.12. Pre-set Protocols Screen. There are Pre-set protocols for bacteria, fungi, and mam-malian cells. Use this screen t

Table of ContentsSection 1 Introduction and Safety Information: The Gene Pulser XcellSystem ...

Pre-set Protocol: CHOFig. 3.14. Protocol Detail Screen for CHO cells in the Pre-set Protocols menu.Table 3.4. Optimized settings found in the Pre-s

3.4.3 Modifying Pre-set Protocol ParametersThe parameters for a Pre-set protocol may be changed as follows.• From the Protocol Detail screen, press t

3.5 User ProtocolsUser Protocols enable users to store their own protocols for the Gene Pulser Xcell. Up to 144electroporation protocols may be progr

27• To enter the User Protocols screen from the Home screen (Figure 3.2):• Press 5 or use the Up and Down Arrow keys to highlight “User Protocols”, th

User Protocols – User Directory Screen (screen 1)User Protocols – User Directory Screen (screen 2)Fig. 3.15. User Directory screen.User Protocols Scr

Select Method ScreenFig. 3.17. Select Method screen.• When “Exponential protocol” is selected, the Exponential Decay Protocol Detail screen appears(F

• When “Square wave protocol” is selected, the Square Wave Protocol Detail screen appears (Figure 3.20) with the User Name on the first line.Square Wa

• Use the Up and Down Arrow keys to highlight the value of the first parameter to be changed. Usethe alpha-numeric keypad to enter a new value. Altern

• Use the alpha-numeric keypad or the Up and Down Arrow keys to select the position with the protocol to be deleted.• Press the Delete key. A warning

Warning screen: delete nameFig. 3.23. Warning screen: delete name.3.5.6 Renaming a User Name or a User Protocol nameRename a User Name or a User Pr

3.5.1 Using a User Protocol (Quick Guide) ...263.5.2 Creating a New User Name ...

3.6 Last PulseFrom the Home screen (Figure 3.2), press 6 or use the Up or Down Arrow keys to select “Last pulse”,then press Enter to display the Prot

Exponential Decay Protocol Optimize ScreenFig. 3.25. Exponential Decay Protocol Optimize screen.Time Constant Protocol Optimize ScreenFig. 3.26. Time

• When the necessary parameter values have been specified, a flashing “P” appears in the characterspace in the lower right corner of the LCD display i

Date ScreenNote: this is the default screen with the default clock preference set as MM/DD/YY; if the userchanges the clock preference to DD/MM/YY, t

The following are examples of data screens. It is not possible to edit a Data screen.Data screen (User Protocol, Square Wave)Data screen (Manual Mode,

3.9 MeasurementsThis program allows the user to measure the resistance of a sample prior to pulsing in the Gene PulserXcell and to measure the values

3.9.2 Calibration and Measurement of Capacitors in the CE ModuleBecause electrolytic capacitors change value slowly over time, to keep the instrument

3.10 User Preferences• From the Home screen, press 10 or use the Up or Down Arrow keys to highlight “User preferences”,then press Enter to display th

• Press the Left or Right Arrow keys to toggle between MM/DD/YY and DD/MM/YY. Press Enter tomake a selection and to bring the cursor to the field belo

• From the User Preferences screen press 3 or use the Up or Down Arrow keys to highlight “Sleepfunction”, then press Enter to select and to view the S

6.3.3 Solutions and Reagents ...576.4 Bacillus cereus...

3.11.2 Pulse Trac Diagnostic AlgorithmThis test algorithm tests and selects the optimal capacitor circuit of the Gene Pulser Xcell unit and theCE Modu

4.1 Exponential Decay PulsesThe exponential decay circuit of the Gene Pulser Xcell generates an electrical pulse by discharging a capacitor.When a ca

The decrease in voltage that occurs with a square wave pulse is inversely related to both the capaci-tance of the instrument and the resistance of the

Table 4.1 Droop associated with pulse length at various sample resistances for thehigh-voltage and low-voltage ranges for the Gene Pulser Xcell.High

Section 5Factors Affecting Electroporation: Optimizing ElectroporationThe electrical conditions for the electroporation of cells have been verified t

5.2 DNAWhile the majority of electroporation applications involve delivery of plasmid DNA to cells, nearly any typeof molecule can be introduced into

The addition of even small concentrations of ionic compounds significantly reduces the resistance ofhigh resistance samples and may cause arcing. Resi

Fig. 5.1. Resistance of solutions (A) NaCl and MgCl2and of (B) buffers of NaPO4at pH 6.1 and 7.3and HEPES at pH 7.5. Resistance was measured in 0.2 c

Table 5.1. Resistance of Water in 0.2 cm Cuvettes to which TE has been added1SAMPLE Rsample(ohms) Rsample(ohms)(40 µl volume) (200 µl volume)Water &g

536. Resuspend the pellet in 250 ml of ice-cold 10% glycerol. Centrifuge at 4000 x g for 15 minutes at4°C; carefully pour off and discard the supernat

8.1.1 Attached Cells...708.1.2 Suspension Cells...

5. SOB: 2.0 g Tryptone peptone, 0.5 g Yeast extract, 0.2 ml 5 M NaCl, 0.25 ml 1 M KCl; dissolvein 90 ml water. Adjust pH to 7.0. Bring volume to 100

4. From the Home screen on Gene Pulser Xcell open the Pre-set Protocols screen, then the BacterialProtocol screen (press 4, then Enter twice). To sele

6.3 Agrobacterium tumefaciens6.3.1 Preparation of Electrocompetent CellsSee Lin (1995) for additional information.Gene Pulser Xcell conditions: C =

8. Incubate the cells 3 hr at 30°C, shaking at 250 rpm. Plate aliquots of the electroporated cells onYM agar plates containing the appropriate selecti

4. From the Home screen on Gene Pulser Xcell open the Pre-set Protocols screen, then the BacterialProtocol screen (press 4, then Enter twice). To sele

9. Discard the supernatant. Resuspend each pellet in 12 ml of sterile, ice cold SMEB buffer. Transferto a chilled 30 ml Oakridge tube.10. Pellet the c

6.6 Streptococcus pyogenes6.6.1 Preparation of Electrocompetent Cells From Simon & Ferretti (1995).Using this method, we have obtained transform

8. Plate onto THY agar plates with selective antibiotic. Incubate 1–2 D at 37°C.6.6.3 Solutions and reagents1. THY media (Todd-Hewitt broth with 0.2%

5. Immediately after the pulse place the cuvette on ice for 30 min. Add 1 ml MRS/SG to the cuvetteand transfer the plasmid/cell suspension to a steril

5. Carefully pour off and discard the supernatant; place the centrifuge bottles with the cell pellets onice. 6. Add 80 ml of sterile water to the bot

Section 1The Gene Pulser Xcell™Electroporation SystemThe Gene Pulser Xcell is a pulse generator that uses capacitors to produce controlled exponential

8. Plate aliquots of the electroporated cells on selective agar plates containing 1 M sorbitol. Incubateplates for 48–72 hrs at 30°C.7.1.3 Solutions

7.2.2 Electroporation1. For each sample to be electroporated, prepare a 17 x 100 mm sterile tube with 1 ml of 1.2 MSorbitol and place on ice; also pla

5. Add 100 ml of sterile, YPD/HEPES to each of the bottles and vortex to resuspend the cell pellets;add 2.5 ml of 1M DTT; mix gently. Incubate the cel

5. 1 M HEPES, pH 8.0: 23.8 g HEPES (MW = 238.3), dissolve in ~65 ml water. Adjust pH to 8.0 with5 N NaOH. Bring volume to 100 ml with water. Steriliz

4. From the Home Screen on Gene Pulser Xcell open the Fungal Protocol Screen (press 4, Enter, 2,Enter). To select C. albicans, press 4 or the Down Arr

5. Carefully pour off and discard the supernatant; place the centrifuge bottles with the cell pellets onice. 6. Pool the cell pellets and resuspend i

Section 8Electroporation of Mammalian CellsThis procedure requires a Gene Pulser Xcell main unit and CE Module.8.1 Preparation of Electrocompetent Ce

2. Add plasmid DNA(s) to the electroporation cuvettes. The amount of DNA required per sample isdependent on the cell type. Start with a concentration

Section 9ReferencesAnderson, M.L.M., Spandidos, D.A., and Coggins, J.R., Electroporation of lymphoid cells: factorsaffecting the efficiency of transfe

Fromm, M., Callis, J., Taylor, L.P., and Walbot, V., “Electroporation of DNA and RNA into plant protoplasts”,in Methods Enzymol., vol. 153, Academic P

There are no user serviceable parts within the unit. The operator should make no attempt to open anycase cover or defeat any safety interlock. This in

Silo-Suh, L.A., Lethbridge, B.J., Raffel, S.J., He, H., Clardy, J., and Hanelsman, J., Biological activitiesof two fungistatic antibiotics produced by

Section 10Specifications and Product Information10.1 System Specifications165-2660 Gene Pulser Xcell Total SystemIncludes Main Unit, CE module, PC mo

10.2 Product InformationCatalogNumber Product Description165-2660 Gene Pulser Xcell Total System for eukaryotic and microbial cells, 100–240 V, 50/60

4006217 Rev ALife ScienceGroupWeb sitewww.bio-rad.com Bio-Rad Laboratories Main OfficeAlso in: AustraliaPh. 02 9914 2800, Fx. 02 9914 2889 AustriaB

Warning: The Gene Pulser Xcell generates, uses, and radiates radio frequency energy. If it is not used inaccordance with the instructions given in thi