Gene Pulser II Instruction Manual 165-xxxx series Rev C

Bio-Rad Laboratories Life Science Group 2000 Alfred Nobel Drive Hercules, California 94547 Telephone (510) 741-1000 Fax: (510) 741-5800 www.bio-rad.com

Bulletin 0000

US/EG

Rev A

Australia, Bio-Rad Laboratories Pty. Ltd., Block Y, Unit 1, Regents Park Industrial Estate, 391 Park Road, Regents Park, NSW 2143 Phone 02 9914 2800 • Fax 02 9914 2889 Austria, Bio-Rad Laboratories Ges.m.b.H., Auhofstraße 78D, A-1130 Wien • Phone (01)-877 89 01 • Fax (01)-876 56 29 Belgium, Bio-Rad Laboratories S.A.-N.V., Begoniastraat 5, B-9810 Nazareth • Phone 09-385 55 11 • Free Phone 0800/97032 • Fax 09-385 65 54 Brazil, Bio-Rad Laboratories (Brazil), Rua dos Invalidos 212 - 5 andar, Lapa - Rio de Janeiro - RJ, CEP 20331-020 • Phone 55 21 507 6191 Canada, Bio-Rad Laboratories (Canada) Ltd., 5671 McAdam Road, Mississauga, Ontario L4Z 1N9 • Phone (905) 712-2771 • Fax (905) 712-2990 China, Bio-Rad China (Beijing), Rm 615, Shang Fang Plaza, No. 27, North Third Round Center Road, West District, Beijing 100029 Phone 86-10-8201-1366/68 • Fax 86-10-8201-1367 Denmark, Bio-Rad Laboratories, Generatorvej 8 C, 2730 Herlev • Phone 45 44 52-1000 • Fax 45 44 52-1001 Finland, Bio-Rad Laboratories, Pihatörmä 1A, FIN-02240 Espoo • Phone 358 (0)9 804 2200 • Fax 358 (0)9 804 1110 France, Bio-Rad Laboratories, 3, Boulevard Raymond Poincaré, 92430 Marnes-la-Coquette • Phone 01 47 95 69 65 • Fax 01 47 41 9133 Germany, Bio-Rad Laboratories GmbH, Heidemannstraße 164, D-80939 München, Postfach 45 01 33, D-80901 München Phone 089 318 84-177 • Fax 089 318 84-123 Hong Kong, Bio-Rad Pacific Ltd., Unit 1111, 11/F, New Kowloon Plaza, 38 Tai Kok Tsui Road, Tai Kok Tsui, Kowloon Phone 852-2789-3300 • Fax 852-2789-1257 India, Bio-Rad Laboratories (India) Pvt. Ltd., B&B1, Enkay Towers Vanijyanikunj, Udhyog Vihar Phase V, Gurgaon, Haryana 122016 Phone (91-124)-6398112/113/114 • Fax (91-124)-6398115 Israel, Bio-Rad Laboratories, Ltd., 14 Homa Street, P.O. Box 5044, Rishon Le Zion 75150 • Phone 03 951 4124 • Fax 03 951 4129 Italy, Bio-Rad Laboratories S.r.l., Via M. Peroglio 23, 00144 Rome • Phone 34 91 590 5200 • Fax 34 91 590 5211 Japan, Nippon Bio-Rad Laboratories KK, 7-18 Higashi-Nippori 5-chome, Arakawa-ku Tokyo 116-0014 • Phone 03-5811-6270 • Fax 03-5811-6272 Korea, Bio-Rad Korea Ltd., Cambridge Building, 1461-15 Seocho-Dong Seocho-Ku, Seoul 137-070 • Phone 82-2-3473-4460 • Fax 82-2-3472-7003 Latin America, Bio-Rad Latin America, 14100 Palmetto Frontage Road, Suite 101, Miami Lakes, Florida USA 33016 • Phone 305-894-5950 • Fax 305-894-5960 Mexico, Bio-Rad Laboratorios Mexico, Adolfo Prieto No. 1653, Col. De Valle, CP. 03100, Mexico D.F. • Phone 52 5 534 2552 to 54 • Fax 52 5 524 5971 The Netherlands, Bio-Rad Laboratories B.V., Fokkerstraat 10, 3905 KV Veenendaal • Phone 0318-540666 • Fax 0318-542216 New Zealand, Bio-Rad Laboratories Pty Ltd., PO Box 300-571, Albany, Auckland • Phone 64-9-4152280 • Fax 64-9-443 3097 Norway, Bio-Rad Laboratories, Johan Scharffenbergs vei 91, N-0694 Oslo • Phone 47-23-38-41-30 • Fax 47-23-38-41-39 Russia, Bio-Rad Laboratorii, ul. Butirskaya 79 "B", office 156 RF-125015 Moscow • Phone 7 095 979 98 00 • Fax 7 095 979 98 56 Singapore, Bio-Rad Laboratories, Singapore, 211 Henderson Rd. #03-02, Henderson Industrial Park, 159552 • Phone 65-2729877 • Fax 65-2734835 Spain, Bio-Rad Laboratories, S.A., Lopez de Hoyos, 245-247, 28043 Madrid • Phone 34-91-590-5200 • Fax 34-91-590-5211 Sweden, Bio-Rad Laboratories AB, Vintergatan 1, Box 1097, S-172 22 Sundbyberg • Phone 46 (0)8-55 51 27 00 • Fax 46 (0)8-55 51 27 80 Switzerland, Bio-Rad Laboratories AG, Nenzlingerweg 2, CH-4153 Reinach • Phone 061-717-9555 • Fax 061-717-9550 United Kingdom, Bio-Rad Laboratories Ltd., Bio-Rad House, Maylands Avenue, Hemel Hempstead, Hertfordshire HP2 7TD Phone 0181 328 2000 • Free Phone 0800-181134 • Fax 01442-259118 00-000

0000

Sig 1200

M1652105 Rev C

Warranty The Gene Pulser II electroporation system is warranted against defects in materials and workmanship for 1 year. If any defects occur in the instruments or accessories during this warranty period, Bio-Rad Laboratories will repair or replace the defective parts at its discretion without charge. The following defects, however, are specifically excluded: 1. Defects caused by improper operation. 2. Repair or modification done by anyone other than Bio-Rad Laboratories or an authorized agent. 3. Damage caused by substituting alternative parts. 4. Use of fittings or spare parts supplied by anyone other than Bio-Rad Laboratories. 5. Damage caused by accident or misuse. 6. Damage caused by disaster. 7. Corrosion caused by improper solvent or sample. This warranty does not apply to parts listed below: Fuses For any inquiry or request for repair service, contact Bio-Rad Laboratories. Inform Bio-Rad of the model and serial number of your instrument. IMPORTANT: This Bio-Rad instrument is designed and certified to meet IEC 1010-1* safety standards. Certified products are safe to use when operated in accordance with the instruction manual. This instrument should not be modified or altered in any way. Alteration of this instrument will: Void the manufacturer’s warranty Void the IEC 1010-1 safety certification Create a potential safety hazard Bio-Rad Laboratories is not responsible for any injury or damage caused by the use of this instrument for purposes other than those for which it is intended, or by modifications of the instrument not performed by Bio-Rad Laboratories or an authorized agent. *IEC 1010-1 is an internationally accepted electrical safety standard for laboratory instruments.

Table of Contents Page

Section 1

Introduction ...1

1.1

Gene Pulser II System Description ...1

Section 2

Unpacking and System Installation...2

2.1 2.2 2.3

Safety ...2 Unpacking The System Components...3 Setting Up The System...3

Section 3

Guide to Switches and Keypads...7

3.1 3.2 3.3 3.4 3.5

Gene Pulser II Unit Control Panel ...8 Pulse Controller II Front Panel ...13 Pulse Controller PLUS Front Panel ...14 Capacitance Extender II Front Panel ...15 Capacitance Extender PLUS Front Panel...16

Section 4

Operation...16

4.1

Instructions for Using the Gene Pulser II unit and the Pulse Controller II (or PLUS) Accessory ...16 Instructions for Using the Gene Pulser II unit with the Capacitance Extender II (or PLUS ) Accessory Module ...17

4.2

Section 5

The Pulse Trac™ system...18

5.1 5.2

Pulse Trac System Description ...18 Pulse Trac Diagnostic Algorithm...18

Section 6

Instrument Diagnostics and Troubleshooting ...20

Section 7

Specifications and Product Information ...21

7.1 7.2

System Specifications ...21 Product Information ...22

Appendix A

Review of Electroporation Fundamentals ...22

i

ii

Section 1 Introduction This manual will familiarize you with the features and the operation of the Gene Pulser II apparatus with its accessory components, the Pulse Controller II or Pulse Controller PLUS module and the Capacitance Extender II or Capacitance Extender PLUS module. Section 2, Unpacking and System Installation, contains important safety information as well as instructions on unpacking and connecting the Gene Pulser instrument to the Pulse Controller II or Pulse Controller PLUS module and the Capacitance Extender II or Capacitance Extender PLUS module for electroporation. Use Section 3, Guide to Switches and Keypads, to learn about the controls and read-out information that the Gene Pulser II apparatus provides with the Pulse Controller II or Pulse Controller PLUS module and Capacitance Extender II or Capacitance Extender PLUS module. In Section 4, Operation, you can examine basic set up and protocols for both prokaryotic and eukaryotic electroporation.

1.1 Gene Pulser II System Description The new Gene Pulser II system* is a completely redesigned electroporation system, which offers improved performance, flexibility, sample protection, and safety. The Gene Pulser II system uses the Pulse Trac™ waveform delivery system to generate the most accurate exponential decay pulses possible for optimal cell transformation within an electroporation cuvette. The Pulse Trac delivery system accurately calculates the time constant of each pulse based on what is actually delivered to the sample. The revolutionary Pulse Trac waveform delivery system provides •

Sample conductivity measurement integrated with pulse output for true waveform delivery regardless of sample

•

Internal calibration and circuit monitoring program for accurate pulse delivery throughout the lifetime of the unit

•

Improved time constant accuracy

Other features of the new Gene Pulser II system include •

Error free assembly, since each module uses unique cable connectors, keyed to fit only in the proper orientation and location

•

Automatic display of time constant after every pulse, to make it easier to record this essential pulse parameter

•

Easier optimization of novel prokaryotic applications with a wider range of high voltage capacitors (1.0 , 3.0, 10, 25, and 50 µF) in the Gene Pulser II unit

•

Pinpointing of optimal field strengths, with voltage selection in 2 V increments for mammalian cell electroporation (low voltage/high capacitance) and operation at a higher maximum voltage (up to 500 V). In addition, voltage selection in 10 V increments for bacterial (high voltage/low capacitance) electroporation

•

Increased control of high voltage pulses used for bacteria and yeast electro-transformation with the five new resistors in the Pulse Controller PLUS module

* United States Patent numbers 4,750,100; 4,910,149 and patents in application.

1

•

Expanded range of precision capacitors for reproducible mammalian cell electroporation; 25 µF–3,275 µF with the Capacitance Extender PLUS module or 25 µF–1,075 µF with the Capacitance Extender II module. This permits greater time constant reproducibility and flexibility with electronically calibrated capacitors in 25 µF increments. At any time, the Pulse Trac circuitry can verify and calibrate these capacitors within ±10% variation for the ultimate in pulse reproducibility and precision.

•

Designed and certified to meet IEC 1010 safety standards

•

Entire system is completely protected from sample arcs, regardless of module or sample used

•

Expanded safety, with charges automatically diverted safely to ground whenever the circuit is interrupted during pulse delivery.

The Gene Pulser II unit may be used with either of two new types of accessory modules. For electroporation of bacteria and yeast, use the Pulse Controller II or Pulse Controller PLUS module. For electroporation of mammalian cells and embryonic tissue, use the Capacitance Extender II or Capacitance Extender PLUS module.

Section 2 Unpacking and System Installation Read this important information before you use the Gene Pulser II electroporation device with the Pulse Controller II or Pulse Controller PLUS module and the Capacitance Extender II or Capacitance Extender PLUS module.

2.1 Safety This instrument is intended for laboratory use only. This product conforms to the “Class A” standards for electromagnetic emissions intended for laboratory equipment applications. It is possible that emissions from this product may interfere with some sensitive appliances when placed nearby or in the same circuit as those appliances. The user should be aware of this potential and take appropriate measures to avoid interference. No part of the Gene Pulser II system should be used if obvious external case damage has occurred or the electronic displays are not functioning as described in the manual. This instrument is only to be used with the components provided (or their authorized additions or replacements) including, but not limited to, supplied cables and shocking chamber. The operating temperature range for the Gene Pulser II system and its associated components is 0–35 °C. This Bio-Rad instrument is designed and certified to meet IEC 1010* safety standards. There are no user serviceable parts within the unit. The operator should make no attempt to open any case cover or defeat any safety interlock. This instrument must not be altered or modified in any way. Alteration of this instrument will •

Void the manufacturer’s warranty

•

Void the IEC 1010 safety certification

•

Create a potential safety hazard

Bio-Rad is not responsible for any injury or damage caused by the use of this instrument for purposes other than those for which it is intended or by modification of the instrument not performed by Bio-Rad or an authorized agent. *IEC 1010 is an internationally accepted electrical safety standard for laboratory instruments.

2

2.2 Unpacking the System Components Remove all packing material and connect components on a flat, dry surface near an appropriate electrical outlet. The Gene Pulser II apparatus should arrive complete with the following components 1

Gene Pulser II unit

1

Gene Pulser II shocking chamber (clear plastic cover with attached red and black cables, base with attached leads, and cuvette holder)

1

Cuvette rack

5

0.1 cm cuvettes

5

0.2 cm cuvettes

5

0.4 cm cuvettes

1

Warranty card (please complete and return)

The Pulse Controller II or Pulse Controller PLUS accessory to the Gene Pulser II unit should arrive complete with the following components 1

Pulse Controller II or Pulse Controller PLUS module (with integrated leads)

1

Warranty card (please complete and return)

The Capacitance Extender II or Capacitance Extender PLUS accessory to the Gene Pulser II unit should arrive complete with the following components 1

Capacitance Extender II or Capacitance Extender PLUS module accessory (with integrated leads)

1

RS 232 readout cable (25 pin)

1

Warranty card (please complete and return)

2.3 Setting Up the System Setting Up the Gene Pulser II Unit To use the Gene Pulser II unit with either the Pulse Controller II or Pulse Controller PLUS and the Capacitance Extender II or Capacitance Extender PLUS, follow this procedure. 1. Verify Gene Pulser II unit voltage compatibility with local electrical output by checking that the red voltage switches in the back of the unit are in the proper position (Figure 2.1). • Units operating in the 110-120 V range must have the red voltage switches positioned so 115 V is visible on both switches. • Units operating in the 220-240 V range must have the red voltage switches positioned so 230 V is visible on both switches. 2. Attach the power cord to the three pronged receptacle on the back of the Gene Pulser II unit (Figure 2.1) and plug the unit into an appropriate electrical outlet or power strip.

3

Power cord receptacle PULSE CONTROLLER

P O W E R

Fuses

CAPACITANCE EXTENDER HIGH-VOLTAGE

Output jack receptacles for Capacitance Extender II (or PLUS) and Pulse Controller II (or PLUS) module

Voltage switches VOLTAGE

SELECT

T O

25-pin Capacitance Extender II (or PLUS) port

P R I N T E R

Fig. 2.1. Rear panel of Gene Pulser II unit. Voltage switches, cable ports, power cord, and output jack receptacles.

3. Assemble the shocking chamber by placing the clear plastic cover (end without cables) over the mid point of the base. Position the plastic cover so that base leads insert securely into the cable receptacles on the plastic cover. Insert the cuvette slide into base (Figure 2.2).

Output jack leads

Cover

Cable receptacles

Base leads

Insert cuvette

Shocking chamber base Cuvette slide

Fig. 2.2. Assembly of Gene Pulser II unit shocking chamber.

4

Connecting the Pulse Controller II or Pulse Controller PLUS Accessory to the Gene Pulser II Unit 1. Place the Pulse Controller II or (PLUS) module on top of or near the Gene Pulser II unit. 2. Insert the red/black leads that are permanently attached to the back of the Pulse Controller II (or Pulse Controller PLUS) into the appropriately labeled receptacle on the back of the Gene Pulser II unit. The leads are keyed so that they will insert only into the proper output jack in the correct red/black orientation.

HIGH-VOLTAGE LEADS CONNECT TO PULSE CONTROLLER HV OUT ON GENE PULSER® II REAR

Output jack leads

Fig. 2.3. Rear view (back panel) of Pulse Controller II (or PLUS ) module.

HIGH-VOLTAGE LEADS DISCONNECT 25-PIN CABLE FROM GENE PULSER II OR CAPACITANCE EXTENDER BEFORE REMOVING CAPACITANCE EXTENDER HV OUT LEADS.

CONNECT TO CAPACITANCE EXTENDER HV OUT ON GENE PULSER® II REAR

CONNECT TO CAPACITANCE EXTENDER CONTROL ON GENE PULSER II REAR.

Pulse Controller II (or PLUS)

P O W E R PULSE CONTROLLER

CAPACITANCE EXTENDER HIGH-VOLTAGE

Pulse Controller II (or PLUS) Output jack leads

VOLTAGE

SELECT

T O P R I N T E R

Fig. 2.4. Red/black leads (back panel) of Pulse Controller II (or PLUS) module, inserted into back panel of Gene Pulser II unit.

3. Connect the leads from the shocking chamber to the output jacks on the front of the Pulse Controller II (or PLUS) accessory module. The leads are keyed so that they will insert only in the correct red/black orientation.

5

Connecting the Capacitance Extender II or Capacitance Extender PLUS to the Gene Pulser II Unit 1. Place the Capacitance Extender II (or PLUS) on top of or near the Gene Pulser II unit. If the Pulse Controller II (or PLUS) module is also to be used, we recommend that you first place the Pulse Controller II (or PLUS) module on top of the Gene Pulser II unit and then place the Capacitance Extender II (or PLUS) module atop the Pulse Controller module. 2. Insert the red/black leads that are permanently attached to the back of the Capacitance Extender II (or PLUS) into the appropriately labeled receptacle on the back of the Gene Pulser II unit. The leads are keyed so that they will insert only into the proper output jack in the correct red/black orientation. 3. Complete the connection of the Capacitance Extender II (or PLUS ) module to the Gene Pulser II unit using the supplied RS 232 cable (25 pin). (Figure 2.5 and 2.6). Make certain that the power switch is off on the Gene Pulser II unit. Insert one end of cable (the end with 25 pins inside) into the appropriate connecting port on back panel of Gene Pulser II unit. Secure cable to port with the two cable attachment screwsfinger tighten with a clockwise motion. Attach the other end of the cable into the 25 pin receptacle on the back of the Capacitance Extender II or Capacitance Extender PLUS unit. Secure cable to port with the two cable attachment screws-finger tighten with a clockwise motion.

HIGH-VOLTAGE LEADS CONNECT TO CAPACITANCE EXTENDER HV OUT ON GENE PULSER® II REAR

DISCONNECT 25-PIN CABLE FROM GENE PULSER II OR CAPACITANCE EXTENDER BEFORE REMOVING CAPACITANCE EXTENDER HV OUT LEADS. CONNECT TO CAPACITANCE EXTENDER CONTROL ON GENE PULSER II REAR.

A

25 pin Capacitance Extender II Port Output jack leads

HIGH-VOLTAGE LEADS

DISCONNECT 25-PIN CABLE FROM GENE PULSER II OR CAPACITANCE EXTENDER BEFORE REMOVING CAPACITANCE EXTENDER HV OUT LEADS.

CONNECT TO CAPACITANCE EXTENDER HV OUT ON GENE PULSER® II REAR

B

CONNECT TO CAPACITANCE EXTENDER CONTROL ON GENE PULSER® II REAR.

Output jack leads 25 pin Capacitance Extender PLUS Port

Fig. 2.5. Back panel of (A) Capacitance Extender II or (B) Capacitance Extender PLUS module.

6

HIGH-VOLTAGE LEADS DISCONNECT 25-PIN CABLE FROM GENE PULSER II OR CAPACITANCE EXTENDER BEFORE REMOVING CAPACITANCE EXTENDER HV OUT LEADS.

CONNECT TO CAPACITANCE EXTENDER HV OUT ON GENE PULSER® II REAR

CONNECT TO CAPACITANCE EXTENDER CONTROL ON GENE PULSER II REAR.

25 pin Capacitance Extender II (or PLUS) Port

P O W E R PULSE CONTROLLER

CAPACITANCE EXTENDER HIGH-VOLTAGE

VOLTAGE

Capacitance Extender II (or PLUS) Output jack leads

SELECT

T O P R I N T E R

Fig. 2.6. Red/black leads and the 25 pin RS232 cable of the Capacitance Extender II (or PLUS) module (back panel) connected to the appropriate output jacks and port on the back panel of the Gene Pulser II unit.

4. When using the Capacitance Extender II (or PLUS ) module, connect the leads from the shocking chamber to the output jacks on the front of the Gene Pulser II unit. The leads are keyed so that they will insert only in the correct red/black orientation.

Section 3 Guide to Switches and Keypads Before operating the Gene Pulser II system with either the Pulse Controller II (or PLUS) or the Capacitance Extender II (or PLUS) accessory, it is helpful to understand the display information, switches, and keypad controls for these devices.

7

3.1 Gene Pulser II Unit Control Panel 2

11

3

GENE PULSER ® II

CAPACITANCE (µF)

10 3.0

2.5

PULSE

1.0 SET VOLTS (kV)

SET HIGH CAP (µF x1000)

ACTUAL VOLTS (kV)

TIME CONST. (msec)

RAISE

LOWER

50

LOW CAP (2500 V MAX.) HIGH CAP (500 V MAX.)

MEASURE CAP. (µF)

SAMPLE RESISTANCE

OUTPUT

HIGH VOLTAGE

1

4

6

7 9

8

5

12

10

Fig. 3.1. The control panel of the Gene Pulser II apparatus. Front panel identification of switches and keypads for the Gene Pulser II unit. Note that the keypad(s) are active when the light(s) above them are illuminated.

1. POWER Function Turns unit on and off

2. PULSE

Result/Display With the power on, the system initializes in the Set Volts mode (the light above SET VOLTS will be illuminated).

PULSE

Function Pressing both Pulse buttons continuously activates the Pulse Trac system to deliver the set voltage to the cuvette via the selected capacitor(s).

Result/Display Capacitor selected is charged with set voltage, and CHg is displayed. Pulse is delivered to cuvette as tone sounds and PLS is displayed. Release the pulse buttons are after the tone sounds. The time constant of the pulse is then automatically displayed.

If the two Measure Cap. keypads are selected, pressing both the Pulse buttons continuously activates the Pulse Trac system to measure the selected capacitor.

The Pulse Trac system is activated to measure the actual storage capacity of selected capacitor. No energy is delivered to the sample cuvette. (You can verify this, as the time constant for this Pulse mode is 0.00). See Measure Cap. key description below.

8

CAPACITANCE (µF)

10 3.0

1.0

2.5

50

LOW CAP (2500 V MAX.)

3. CAPACITANCE (µF) rotary switch

HIGH CAP (500 V MAX.)

Function LOW CAP. (2,500 V max.). Positions 1.0, 3.0, 10, 25, or 50 µF select the specific high voltage/low value capacitor inside the Gene Pulser II unit.

Result/Display Selecting any of the Low Cap. capacitors (used with the Pulse Controller II (or PLUS) module-high voltage) allows the Set Volts adjustment to range from 0.2 to 2.5 kV in 10 V (0.01 kV) increments.

HIGH CAP. (500 V max.) position activates low voltage circuitry for use of higher value capacitors in Capacitance Extender II or PLUS accessory module.

Selecting the High Cap. position on rotary Capacitance switch activates the Capacitance Extender II (or PLUS) module and allows the Set Volts adjustment to range from 0.050 to 0.500 kV (50 to 500 V) with 2 V (0.002 kV) increments.

SET VOLTS (kV)

4. SET VOLTS

RAISE

LOWER

then 5. RAISE LOWER

Function Result/Display Pressing SET VOLTS • The Set Volts display is in kilovolts. activates the Raise and Lower keypads that are used to adjust the desired voltage to be delivered to the sample cuvette. To Set volts for High voltage/low capacitance Pulse Trac waveforms (using Pulse Controller II or PLUS module).

High voltage/low capacitance Pulse Trac waveforms: • The 3 digit LED display reads 0.00 when any of the Low Cap. (1, 3, 10, 25, or 50 µF) capacitors are selected for use in delivering high voltage. The Set Volts adjustment ranges from 0.2 to 2.5 kV, in 10 volt (0.01 kV) increments when any of these high voltage capacitors are selected. • Pre-set high voltage settings used with the Pulse Controller II (or PLUS) module are available by pressing both the Raise and Lower keypads simultaneously. Pressing these two keys once will set the voltage to 1.8 kV (1.80 will be displayed). Pressing both the Raise and Lower keypads twice will set the voltage to 2.5 kV and 2.50 will be displayed. Pressing both buttons a third time will reset the voltage to 0.00 kV.

9

Function To Set volts for Low voltage/high capacitance Pulse Trac waveforms (using Capacitance Extender II or PLUS module).

Result/Display Low voltage/high capacitance Pulse Trac waveforms: The 3 digit LED display reads .000 if the High Cap. position is selected. The Set Volts adjustment ranges from 0.050 to 0.500 kV (50 to 500 V) with 2 V (0.002 kV) increments when High Cap. position is selected on rotary Capacitance switch. [Note: If the voltage is set at 0.00, the display will read no if the pulse buttons are pressed]. Pre-set low voltage settings used with the Capacitance Extender II (or PLUS) module are available by pressing both the Raise and Lower keypads simultaneously. Pressing these two keys once will set the voltage to 0.360 kV (and 0.360 will be displayed). Pressing both the Raise and Lower keypads twice will set the voltage to 0.500 kV and .500 will be displayed. Pressing both buttons a third time will reset the voltage to .000 kV.

SET HIGH CAP (µF x1000)

6. SET HIGH CAP. (µF x 1,000) RAISE

LOWER

then 5. RAISE/LOWER Function Allows Raise and Lower keypads to adjust the selection of capacitors of the Capacitance Extender II (or PLUS) module.

Result/Display When the power is turned on, the capacitor selected is 0.025 (x 1,000) or 25 µF. The display reads .025. Adjustments can be made in 25 µF increments using the Raise or Lower buttons. Choose capacitors between 25 to 1,075 µF [displayed as .025 to 1.07 (x 1,000) µF] in the Capacitance Extender II module or 25 to 3,275 µF [displayed as .025 to 3.27 (x 1,000) µF] in the Capacitance Extender PLUS module. The LED abbreviates the display of capacitors larger than 975 µF as 1.00=1.000, 1.02 =1.025, 1.05 =1.050, 1.07 =1.075 (x 1,000) µF, etc., but actual capacitor values are as cited. Dashes (– – –) will appear on LED display if Capacitance rotary switch is not set on High Cap. position. If the Capacitance Extender II (or PLUS) module is not connected to the Gene Pulser II unit, no will be displayed.

10

ACTUAL VOLTS (kV)

7. ACTUAL VOLTS (kV) Function After pulse, displays actual volts delivered to sample in cuvette using the Pulse Trac system.

Result/Display The Actual Volts (kV) function displays the actual volts delivered to the cuvette. The value is retained until next pulse is delivered and power remains on. Raise and Lower keys cannot be used when the Actual Volts key is active.

TIME CONST. (msec)

8. TIME CONST. (msec) Function Displays the actual measured time constant of the Pulse Trac waveform delivered by Gene Pulser II unit to the cuvette sample. The time constant (τ) is the amount of time required for the actual voltage of the delivered pulse to decrease to a value of 1/e of the true peak pulse. The display is in msec.

Result/Display The actual time constant is automatically displayed as a LED value after a pulse is delivered. This value is retained until next pulse is delivered and power remains ON. The time constant range for high voltage pulses with the Pulse Controller II (or PLUS) module is 0.02 to 80 msec with a 0.02 msec precision. The time constant range for low voltage pulses with the Capacitance Extender II (or PLUS) module is 0.1 msec to 6.5 sec. Time constants larger than 999 msec are displayed in abbreviated form. For example, 1,000 msec is displayed as 1.0t (or 1 x 1,000 msec). Raise and Lower keys cannot be used when the Time Const. key is active.

11

SET HIGH CAP (µF x1000)

MEASURE CAP. (µF)

9. MEASURE CAP (µF) Function Two keypads and Pulse buttons work together to activate the Pulse Trac system to measure actual storage capacity of selected capacitor in µF.

ACTUAL VOLTS (kV)

then 2. PULSE

PULSE

Result/Display When the desired capacitor is selected (using the Capacitance switch or the Set High Cap. keypad), the two Measure Cap keypads are pressed (the two LEDs over the keypads are lit). The Pulse Trac system is activated by pressing both Pulse buttons. Hold both Pulse buttons until tone sounds. Release Pulse buttons. Display first shows CHg, then tst, then capacitor test value. Both Measure Cap. keypads, then both Pulse buttons, must be pressed simultaneously for this function to operate. Display is retained until power switched off or another Measure Cap. test is completed. Display value for capacitors larger than 975 µF is truncated (i.e. 1,000 µF=1.00). No energy is delivered to sample cuvette. (You can verify this, as the time constant for the Pulse Trac mode is 0.00). High voltage capacitor tolerance is ±10%. Low voltage capacitor tolerance is ±20%. (Other electroporation devices have variation standards that approach ±40% that can greatly ACTUAL VOLTS (kV)

10. SAMPLE RESISTANCE Function Activates the Pulse Trac system to monitor sample resistance (includes resistance in Pulse Controller II or PLUS module, if connected).

TIME CONST. (msec)

SAMPLE RESISTANCE

Result/Display Pressing these two keypads allows the unique Pulse Trac system to accurately monitor resistance of its internal arc protection circuitry, depending on the sample conductivity. Value displayed is derived from low current sine-waves into the sample (voltage well below 1 V). The display range is 5–999 ohms when the High Voltage/Low Capacitor mode is used. Use this to verify Pulse Controller II or PLUS module resistor settings. The range is 5–800 ohms when the Low Voltage/High Capacitor mode is used. Use this to estimate changes in sample conductivity when using the Capacitance Extender II (or PLUS) module. When the maximum value for the given resistance range is displayed, the actual resistance may be greater than or equal to the displayed value. Raise and Lower keys cannot be used when these two keys are active.

12

11. LED Function LED (Light Emitting Diode) display

Result/Display Displays Pulse Trac values and function indicators (3 digit).

OUTPUT

12. Shocking Chamber Output Jack

HIGH VOLTAGE

Function Result/Display Shocking chamber output jack Used as receptacle for red/black leads of shocking chamber when the Capacitance Extender II or PLUS module is used for electroporation.

3.2 Pulse Controller II Front Panel 1

2

600

PULSE CONTROLLER II

400

200

800

1000

100

OUTPUT

HIGH VOLTAGE

RESISTANCE (ohmns)

Fig. 3.2. The front control panel of the Pulse Controller II apparatus. Resistor selection switch (1) and shocking chamber output jack (2). 600 400

200

800

1000

100

1. Resistance Selection Switch (ohms) Function Rotary switch selects one of six parallel resistors (100, 200, 400, 600, 800, 1,000 ohm ) for Pulse Trac voltage delivery to sample cuvette.

RESISTANCE (ohmns)

Result/Display Parallel resistors divert current (not voltage) from cuvette sample in Pulse Trac circuit. Used in high voltage electroporation to prevent high current from adversely affecting biological sample during electroporation.

13

Function

Result/Display

Selection of infinity (∞) position engages only 20 ohm protective series resistor and no parallel resistors in the Pulse Trac circuit.

Use the infinity (∞) position when you wish to use the Capacitance Extender II (or PLUS) module without disconnecting the Pulse Controller II (or PLUS) module. Display reads 999 for infinity (∞) position when the Low Caps. setting is used and 800 when the High Caps. setting is used.

OUTPUT

2. Shocking chamber output jack

HIGH VOLTAGE

Function

Result/Display

Connection point for leads from shocking chamber for high voltage (bacterial) electroporation.

Attaching the shocking chamber to Pulse Controller PLUS output jack gives increased circuitry protection to both the sample cuvette and the Gene Pulser II unit in case of an arc.

3.3 Pulse Controller PLUS Front Panel 1

2

300

PULSE CONTROLLER PLUS

200

700 400

600

TO USE HIGH RANGE

100

500

50 LOW RANGE

800

900

1000 RESISTANCE (ohmns)

OUTPUT

HIGH VOLTAGE

HIGH RANGE

Fig. 3.3. The front control panel of the Pulse Controller PLUS apparatus. Resistor selection switches (1) and shocking chamber output jack (2).

300 200

Function Two rotary switches work together to select one of eleven parallel resistors (50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000 ohms) for pulse delivery to sample cuvette.

600

TO USE HIGH RANGE

100

1. Low Range and High Range Resistance Selection Switches (ohms)

700 400

50 LOW RANGE

800

500

900

1000 RESISTANCE (ohmns)

HIGH RANGE

Result/Display Parallel resistors divert current (not voltage) from cuvette sample in Pulse Trac circuit. Used in high voltage electroporation to prevent high current from adversely affecting biological sample during electroporation.

14

Function

Result/Display

(a) Left-most Low Range resistance switch dominates and selects one of five Low Range resistors (50, 100, 200, 300, 400 ohms) or activates the High Range resistance switch settings. To use High Range resistors, set Low Range switch at To Use High Range position, then using right rotary switch, select one of six parallel resistors (500, 600, 700, 800, 900, 1,000 ohms) for engaging parallel resistor in Pulse Trac waveform delivery to sample cuvette. Selection of infinity (∞) position engages no parallel resistors in the Pulse Trac circuit. Only the 20 ohm protective series resistor is engaged.

Use the infinity (∞) position when you wish to use the Capacitance Extender II (or PLUS) module without disconnecting the Pulse Controller II (or PLUS) module. Make certain that both switches are properly positioned. Display reads 999 for infinity (∞) position when Low Caps. setting is used and 800 when High Caps. setting is used.

OUTPUT

2. Shocking chamber output jack Function Connection point for leads from shocking chamber for high voltage (bacterial) electroporation.

HIGH VOLTAGE

Result/Display Attaching the shocking chamber to Pulse Controller PLUS output jack gives increased circuitry protection to both the sample cuvette and the Gene Pulser II unit in case of an arc.

3.4 Capacitance Extender II Front Panel CAPACITANCE EXTENDER II

Fig. 3.4. Front panel of Capacitance Extender II. Note all capacitors are selected from Gene Pulser II unit front panel controls.

15