CLB-Transfection Manual

Contents
1.0 Introduction 2.0 Related Products 3.0 Operating Instructions
3.1 3.2 3.3 3.4 3.5 3.6 3.7 Restrictions Maintenance Safety Instructions Please Read Carefully Waste Disposal CLB-Transfection Device Components Setup Instructions Instructions for Using the CLB-Transfection Device 3.7.1 Pulse Selection 3.7.2 Pulse Execution Service Chipcard Slot

3 4 5
5 5 6 7 7 7 8 8 8 8

3.8

4.0 Transfection Procedure

4.1 4.2 4.3 Transfection of Suspension Cells Using the CLB-Transfection System Transfection of Adherent Cells Using the CLB-Transfection System Optimization of Transfection Conditions

9
9 10 11

Contents

5.0 CLB-Transfection Cell List 6.0 Transformation of Bacteria 7.0 Troubleshooting 8.0 CLB-Transfection Error Codes 9.0 Appendix

12 14 15 17 18

1.0 Introduction

Lonza, the worlds leading supplier in primary cells and leader in transfection technologies, introduces the CLB-Transfection System, designed for highly reproducible and efficient transfection of adherent and suspension cell lines. Reproducible transfection of adherent and suspension cell lines The CLB-Transfection System has been successfully used for high performance transfections of more than 60 cell lines (see Chapter 5). In most cases, it provides efficiencies and viabilities in the range of 50 90%. Easy optimization of your cell line of choice The CLB-Transfection System offers a convenient, easy-to-follow optimization strategy. With the concept of one universal Transfection Buffer that is applied in combination with ten different pulses, optimization of a new cell line is easily achieved and will usually yield satisfactory transfection efficiencies and viabilities.

Transfection of any substrate The CLB-Transfection System offers high flexibility with respect to applications. The same CLB-Transfection Pulse applies for substrates such as DNA, siRNA and mRNA. It is the ideal tool for overexpression studies, gene silencing approaches, protein expression, generation of stable clones and many other applications. Components of the CLB-Transfection System The CLB-Transfection System is based on two unique components: The CLB-Transfection Device that delivers specifically developed electrical pulses and the CLB-Transfection Kit that contains the unique CLB-Transfection Buffer. The CLB-Transfection Kit is available separately. Transformation of bacteria In addition to eukaryotic cell line transfections, the CLB-Transfection Device offers preset pulses for transformation of different bacterial strains.

Introduction 1.0

2.0 Related Products

Cat. No. AWC-1002 VECA-1001 VKA-1001 LT07-218 LT07-121 LT07-117 LT07-115 VDF-1011 VDF-1012 VDF-1013 VDF-1021 VDF-1022 VDF-1023 VDF-1031 VDF-1032 VDF-1033 VDC-1040 Product CLB-Transfection Device Guarantee - 2 year extension CLB-Transfection Kit Electroporation cuvettes for bacterial, 1 mm gap MycoAlert Mycoplasma Detection Kit ViaLight Plus Cell Proliferation and Cytotoxicity BioAssay Kit ToxiLight Non-destructive Cytotoxicity BioAssay Kit ApoGlow Rapid Apoptosis Screening Kit pmaxFP-Green-C Vector pmaxFP-Green-N Vector pmaxFP-Green-PRL Vector pmaxFP-Yellow-C Vector pmaxFP-Yellow-N Vector pmaxFP-Yellow-PRL Vector pmaxFP-Red-C Vector pmaxFP-Red-N Vector pmaxFP-Red-PRL Vector pmaxCloning Vector 50 reactions 50 cuvettes 25 tests 1000 tests 1000 tests 200 tests 20 g 20 g 20 g 20 g 20 g 20 g 20 g 20 g 20 g 20 g Size

3.0 Operating Instructions

3.1 Restrictions 3.2 Maintenance

Medical use restrictions The CLB-Transfection System is intended for research and investigational use by professionals only. Please note that Lonzas CLB-Transfection System is not intended to be used for diagnostic purposes or for testing or treatment in humans. License statement Lonza Cologne AG is holder of various patents, patent applications, copyrights and technical and scientific experience with respect to the CLB-Transfection Technology. Use of Lonzas CLB-Transfection Technology and/or related software requires a license from Lonza Cologne AG. Purchasers are granted a non-exclusive, non-transferable license for a limited use of Lonzas CLB-Transfection Technology and related software for research purposes, the terms of which are disclosed in detail in the license agreement accompanying the shipped CLB-Transfection Device. For license information, kindly contact Lonza Cologne AG by phone +49 (0)221-99199-0 or e-mail ip.cologne@lonza.com.

The CLB-Transfection Device requires very little maintenance for reliable operation. To clean and disinfect the case, first unplug the power supply. Use a damp cloth to wipe down the outer case (water or 70 80% ethanol). Avoid wetting the cuvette holder within the cuvette carousel and the connectors located on the rear of the device. The CLB-Transfection Device has been designed for use under a sterile hood, with or without UV radiation source. Prolonged exposure of the outer casing to UV light will lead to discoloration with no functional impairment of the CLB-Transfection Device. When laminar flows are sterilized on a regular basis by UV radiation overnight, we recommend protection of the device by appropriate shielding or removal during extended UV exposure. The CLB-Transfection Device is protected by two main fuses. Both are inside a receptacle incorporated in the inner power socket (see Fig. 2, page 7). In case of a blown fuse, you can easily replace it. Disconnect the CLB-Transfection Device from power and insert a small flat screwdriver into the slot on the right-hand side of the receptacle to pull it open. To function, both the upper and lower part of the receptable must hold working fuses in the inner positions. Blown fuses can usually be identified by molten interrupted wires inside the glass tube. Only use T630mA or L250V fuses to substitute blown fuses.

Operating Instructions 3.0

3.3

Safety Instructions Please Read Carefully

Use the device with Lonzas certified CLB-Transfection Buffer and Lonzas certified cuvettes only. Use of any other buffer or cuvettes from any other source than Lonza will preclude all warranty and liability claims. Unpack the cuvettes just prior to the experiment. Make sure that the outer contact areas are dry. If any fluid has been spilled in the close vicinity of the CLB-Transfection Device, safety may be compromised. Ensure that no fluid has been in contact with or entered the device. If any fluid has been spilled on the device, the safety may be compromised. To confirm that the use of the device is safe, contact Lonza Scientific Support for actions or precautions. Never place any foreign object on the device to avoid the risk of equipment damage. Do not enter or place foreign objects in the carousel area of the CLB-Transfection Device. If any foreign object has entered the CLB-Transfection Device, safety may be compromised. To confirm that the use of the device is safe, contact Lonza Scientific Support for actions or precautions. If the CLB-Transfection Device has been damaged, ensure that the device can not be used by any personnel and contact Lonza Scientific Support for assistance. All service shall be performed by Lonza authorized personnel only. Handling of device parts that have the possible risk of sample contamination shall always be performed with protective gloves and any disposal of such parts must be according to federal, state or local procedures for clinical waste handling and disposal. Use secure leak proof containers and avoid unprotected handling of such parts.

This symbol means that there is a risk of electric shock. An electric shock could cause death or personal injury. The CLB-Transfection Device has been certified by international safety standards and is safe to use when operated in accordance with this manual. This device is designed to deliver variable high voltage electrical impulses for the purpose of introducing nucleic acids into eukaryotic and prokaryotic cells. These electrical impulses can be DEADLY! Therefore, use this device with care and take the following precautions: Only use the device once you have read and understood the CLB-Transfection Manual. The manual should be accessible for all users. Make sure that each potential user reads and understands it. Do not open the device. The device does not contain userserviceable parts. Under no circumstances should circuit components be interfered with, as they can deliver an electric shock even when system is not in operation. Do not alter the device in any manner. Only use the device when it is set on top of a safe, plain and stable table or bench. Place the device such that easy removal of the power cord is possible at any time. Do not expose the device to a humid environment. The device shall not be exposed to direct sunlight nor be placed in a hot environment. The device is not approved for use in fire or explosion endangered areas, nor for use with inflammable or explosive media. Employ precautions against great impact and vibration in moving and transporting the CLB-Transfection Device.

Lonza Cologne AG disclaims all warranties and shall in no event be liable for any kind of damages caused by or arising out of any operation or use in violation of the above safety and handling instructions.

3.4

Waste Disposal
Rear side of the CLB-Transfection Device Figure 2

Disposal of consumables from CLB-Transfection Kits (cuvettes and CLB-Transfection Buffer): Discard cuvettes and expired CLB-Transfection Buffer residuals in a biohazard container. Refer to your local waste management organization and to the relevant laboratory safety instructions for proper disposal practices.

3.5

CLB-Transfection Device Components

10 9 Power cord receptacle 10 Spare fuse 9

The CLB-Transfection Device is delivered with the following components: 1 CLB-Transfection Device 1 Power cord CLB-Transfection Manual Front panel of the CLB-Transfection Device Figure 1 2 3 6

3.6
1. 2.

Setup Instructions

4 1 1 2 3 4 5 6 7 8 8 5 7

Remove all packing material. Attach the power cord to the receptacle on the rear side of the CLB-Transfection Device (9) and plug it into an appropriate electrical outlet. The device will automatically be in the standby mode. Turn on the device by pressing the power button in the upper left corner of the front panel (1). The device performs a self test accompanied by the Lonza logo and an hour glass in the front display (2). After powering up the first time, the display shows Cell 1.

Power button Display Up button Down button Start button Cuvette carousel Cuvette holder Service Chipcard Slot

Operating Instructions 3.0

3.7

Instructions for Using the CLB-Transfection Device

Pulse Selection

2.

Press the Start button (5) to acknowledge. The device can be re-used immediately after the last pulse execution. In case the device was unable to execute a pulse or the pulse was not completed successfully, the display will indicate the error code. For more information on specific errors, see Chapter 8. To continue working, press the Start button (5) to acknowledge the error message. The device can then be re-used immediately. When you have finished working, please remove the last cuvette from the cuvette holder (7) and switch off the CLB-Transfection Device by pressing the power button (1).

3. 7.1
1.

2.

Turn on the CLB-Transfection Device by using the power button (1). The device performs a self test accompanied by the Lonza logo and an hour glass in the front display (2). After powering up, the display shows a CLB-Transfection Pulse (e.g. Cell 1). Once the display shows a CLB-Transfection Pulse, choose the appropriate pulse by using the Up and Down buttons (3, 4). With all subsequent activations of the CLB-Transfection Device, the display will show the pulse last entered). Your CLB-Transfection Device is now ready for pulse execution.

3.8

Service Chipcard Slot

3.7.2
1.

Pulse Execution

The Service Chipcard Slot for software reinstallations shall be used by Lonza authorized personnel only.

1. After you have chosen the correct pulse, place the closed cuvette filled with the electroporation sample in the cuvette holder (7). Rotate the cuvette carousel (6) 180 clockwise. The carousel must be turned completely to the blocked position in order for the cuvette to contact the electrodes. For pulse execution, press the Start button (5). OK will appear on the display, followed by a beep, if the pulse has been successfully completed. After addition of pre-warmed medium (see Chapter 4 for details), remove the electroporation sample from the cuvette. Discard cuvettes after one use.

Note Only use Lonzas certified cuvettes, which have passed rigorous quality tests. Use of both uncertified cuvettes and re-use of Lonzas certified cuvettes (after even one use) impairs experimental results and proper function of the device and risks damaging of the CLB-Transfection Device itself.

4.0 Transfection Procedure

The following procedure is a general guideline for transfection of adherent and suspension cell lines using the CLB-Transfection System. In order to select the appropriate CLB-Transfection Pulse for your specific cell line, refer to the Cell list (Chapter 4.) within this manual or our website for a regularly updated version (www. Lonza.com/CLB-Transfection). In case your cell line of interest is not listed, please follow the optimization recommendations in Chapter 5. For transformation of bacteria, please see Chapter 6. Materials provided in the CLB-Transfection Kit: (Cat. No. : VECA-1001) 4.5 ml CLB-Transfection Buffer 1 ml CLB-Transfection Supplement 30 g pmaxGFP Vector 50 certified cuvettes Size: The CLB-Transfection Kit is sufficient for 50 transfections. Storage and stability: Store CLB-Transfection Buffer and CLB-Transfection Supplement at 4C. The expiration date is printed on the CLB-Transfection Buffer flask. Once the CLB-Transfection Supplement is added to the CLB-Transfection Buffer, it is stable for three months at 4C. Required materials not provided in CLB-Transfection Kit: Substrate of interest Tissue culture quality 12-well plates (suspension cells) or 6-well plates (adherent cells) PBS For detaching cells: 0.5 mg/ml Trypsin and 0.2 mg/ml EDTA in PBS and supplemented culture media or PBS/0.5% BSA Culture medium containing serum/supplements (freshly prepared); for detailed recommendations, please refer to the respective suppliers instructions Prewarm and equilibrate appropriate volume of culture medium to 37C (1.5 ml per transfection sample) Appropriate number of cells (1 106 5 106 cells per transfection sample)

4.1

Transfection of Suspension Cells Using the CLB-Transfection System

Preparation of the CLB-Transfection Buffer Add 1 ml CLB-Transfection Supplement to 4.5 ml CLB-Transfection Buffer and mix gently. Note Please make sure that the entire CLB-Transfection Supplement is added to the CLB-Transfection Buffer. The ratio of CLB-Transfection Buffer to CLB-Transfection Supplement is 4.5 : 1. For a single reaction use 82 l of CLB-Transfection Buffer plus 18 l of CLB-Transfection Supplement to make 100 l of total reaction volume. One transfection sample contains: 1 106 5 106 cells 1 5 g plasmid DNA (in 1 5 l H2O or TE) or 30 300nM siRNA (3 30 pmol/sample) 100 l CLB-Transfection Buffer 1. Prepare 12-well plates by filling the appropriate number of wells with 1 ml of supplemented culture medium and preincubate/equilibrate plates in a humidified 37C/5% CO2 incubator. Count an aliquot of the cells and determine cell density. Centrifuge the required number of cells (1 106 5 106 cells per transfection sample) at 90xg for 10 minutes at room temperature. Remove supernatant completely. Resuspend the cell pellet carefully in 100 l room temperature CLB-Transfection Buffer per sample. Note: Avoid leaving the cells in CLB-Transfection Buffer for extended periods of time (longer than 15 minutes), as this may reduce cell viability and gene transfer efficiency. Combine 100 l of cell suspension with 1 5 g DNA or 30nM 300nM siRNA (3 30pmol/sample) or other substrates.

2. 3.

4.

5.

Electroporation Procedure 4.0

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6.

Transfer cell/DNA suspension into certified cuvette (sample must cover the bottom of the cuvette without air bubbles). Close the cuvette with the cap. 7. Insert the cuvette with cell/DNA suspension into the cuvette holder and rotate the carousel clockwise to the final position. Select the appropriate CLB-Transfection Pulse (for details see Chapter 5) and apply the selected pulse by pressing the Start button. 8. Take the cuvette out of the holder once the pulse is finished. 9. Immediately add 500 l of the pre-equilibrated culture medium* to the cuvette and gently transfer the sample into the prepared 12-well plates (final volume 1.5 ml media per well). 10. Press the Start button (5) to confirm the notification by the CLB-Transfection Device. 11. Incubate the cells in humidified 37C/5% CO2 incubator until analysis. Gene expression or down regulation, respectively, is often detectable after only 4 8 hours.
*If the cells grow in Dulbeccos Modified Eagle Medium (DMEM) or Minimum Essential Medium (MEM), we recommend using RPMI containing serum/ supplements.

1.

2. 3. 4.

5. 6.

7.

8. 9.

10.

4.2

Transfection of Adherent Cells Using the CLB-Transfection System

11. 12.

Preparation of the CLB-Transfection Buffer Add 1 ml CLB-Transfection Supplement to 4.5 ml CLB-Transfection Buffer and mix gently. The CLB-Transfection Buffer is now ready to use and is stable for 3 months at 4C. Note date of addition on the vial. Note Please make sure that the entire CLB-Transfection Supplement is added to the CLB-Transfection Buffer. The ratio of CLB-Transfection Buffer to CLB-Transfection Supplement is 4.5 : 1. For a single reaction, use 82 l of CLB-Transfection Buffer plus 18 l of CLB-Transfection Supplement to make 100 l of total reaction volume. One transfection sample contains: 1 106 5 106 cells 1 5 g plasmid DNA (in 1 5 l H2O or TE) or 30 300nM siRNA (3 30 pmol/sample) 100 l CLB-Transfection Buffer

13. 14.

Prepare 6-well plates by filling the appropriate number of wells with 1 ml of supplemented culture medium and pre-incubate/ equilibrate plates in a humidified 37C/5% CO2 incubator. Remove media from the cultured cells and wash cells once with PBS. Use at least same volume of PBS as culture media. For harvesting, incubate the cells ~5 minutes at 37C with 0.5 mg/ml Trypsin and 0.2 mg/ml EDTA. Neutralize trypsinization reaction with supplemented culture medium or PBS/0.5% BSA once the majority of the cells (>90%) have been detached. Count an aliquot of the cells and determine cell density. Centrifuge the required number of cells (1 106 5 106 cells per transfection sample) at 90xg for 10 minutes at room temperature. Remove supernatant completely. Resuspend the cell pellet carefully in 100 l room temperature CLB-Transfection Buffer per sample. Note: Avoid leaving the cells in CLB-Transfection Buffer for extended periods of time (longer than 15 minutes), as this may reduce cell viability and gene transfer efficiency. Combine 100 l of cell suspension with 1 5 g DNA or 30nM 300nM siRNA (3 30pmol/sample) or other substrates. Transfer cell/DNA suspension into certified cuvette (sample must cover the bottom of the cuvette without air bubbles). Close the cuvette with the cap. Insert the cuvette with cell/DNA suspension into the cuvette holder and rotate the carousel clockwise to the final position. Select the appropriate CLB-Transfection Pulse (for details see Chapter 5) and apply the selected pulse by pressing the Start button. Take the cuvette out of the holder once the pulse is finished. Immediately add 500 l of the pre-equilibrated culture medium* to the cuvette and gently transfer the sample into the prepared 6-well plates (final volume 1.5 ml media per well). Press the Start button (5) to confirm the notification by the CLB-Transfection Device. Incubate the cells in humidified 37C/5% CO2 incubator until analysis. Gene expression or down regulation, respectively, is often detectable after only 4 8 hours.

*If the cells grow in Dulbeccos Modified Eagle Medium (DMEM) or Minimum Essential Medium (MEM), we recommend using RPMI containing serum/ supplements.

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4.3

Optimization of Transfection Conditions

to CELL 10 (strongest pulse) subsequently in order to find out the most appropriate conditions for your cell line of interest. We recommend using the pmaxGFP Vector for the optimization procedure.

In case your cell line of interest is not listed in the CLB-Transfection Cell List, we recommend performing a simple optimization of the transfection conditions. Please perform 10 different samples (plus one control with untransfected cells) by trying pulses CELL 1 (weakest pulse)

Well 1 2 3 4 5 6 7 8 9 10 11

Pulse CELL 1 CELL 2 CELL 3 CELL 4 CELL 5 CELL 6 CELL 7 CELL 8 CELL 9 CELL 10 No pulse

Transfection Efficiency

Viability

Depending on your experimental needs, choose the pulse which provides you the highest transfection efficiency and or highest viability for further transfections.

Electroporation Procedure 4.0

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5.0 CLB-Transfection Cell List

The CLB-Transfection Cell List provides an overview about transfection conditions for a huge number of cell lines.
Optimal CLB-Transfection Pulse CELL 7 CELL 5 CELL 8 CELL 9 CELL 10 CELL 9 CELL 9 CELL 7 CELL 1 CELL 9 CELL 9 CELL 9 CELL 9 CELL 9 CELL 9 CELL 9 CELL 5 CELL 4 CELL 9 CELL 8 CELL 8 CELL 9 CELL 8 CELL 10 CELL 10 CELL 7 CELL 9 CELL 8 CELL 10 Cell Line 293 32D 3T3-L1 A2058 A-431 A-549 A7r5 AGS B16-F10 BA/F3 BJ CCRF-CEM CHO-K1 COS-1 COS-7 FDC-P1 HCT 116 HeLa HeLa S3 Hep G2 HL-60 HT-1080 HT-29 HuT 78 HUV-EC-C IMR-90 Jurkat (Clone E6-1) K-562 KG-1 Clone # ATCC CRL-1573 ATCC CRL-11346 ATCC CL-173 ATCC CRL-11147 ATCC CRL-1555 ATCC CCL-185 ATCC CRL-1444 ATCC CRL-1739 ATCC CRL-6475 DSMZ ACC 300 ATCC CRL-2522 ATCC CCL-119 ATCC CCL-61 ATCC CRL-1650 ATCC CRL-1651 ATCC CRL-12103 ATCC CCL-247 ATCC CCL-2 ATCC CCL-2.2 ATCC HB-8065 ATCC CCL-240 ATCC CCL-121 ATCC HTB-38 ATCC TIB-161 ATCC CRL-1730 ATCC CCL-186 ATCC TIB-152 ATCC CCL-243 ATCC CCL-246 Transfection Efficiency (%) 97 76 50 75 54 70 57 58 78 90 52 59 84 83 98 58 78 85 54 50 61 86 50 53 48 53 52 86 70 Viability (%) 86 66 70 48 62 70 60 63 79 61 76 50 91 52 95 55 60 84 80 95 74 75 51 64 61 54 75 67 84

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Cell Line KG-1a L428 L6 LNCaP (Clone FGC) MCF7 MDA-MB-453 MDA-MB-468 MDBK MDCK (NBL-2) MDCK II MOLT-4 NCI-H1299 Neuro-2a NIH/3T3 NIH:OVCAR-3 NK-92 NRK NS0 P815 PANC-1 PC-12 Raji Ramos (RA 1) RAW 264.7 RBL-2H3 S49 SK-N-SH SK-OV-3 T/G HA-VSMC THP-1 U-2 OS U-937 Vero WI-38

Clone # ATCC CCL-246.1 DSMZ ACC 197 ATCC CRL-1458 ATCC CRL-1740 ATCC HTB-22 ATCC HTB-131 ATCC HTB-132 ATCC CCL-22 ATCC CCL-34 ECACC 00062107 ATCC CRL-1582 ATCC CCL-246.1 ATCC CCL-131 ATCC CRL-1658 ATCC HTB-161 ATCC CRL-2407 ATCC CRL-6509 ECACC 85110503 ATCC TIB-64 ATCC CRL-1469 ATCC CRL-1721 ATCC CCL-86 ATCC CRL-1596 ATCC TIB-71 ATCC CRL-2256 ATCC TIB-36 ATCC HTB-11 ATCC HTB-77 ATCC CRL-1999 ATCC TIB-202 ATCC HTB-96 ATCC CRL-1593.2 ATCC CCL-81 ATCC CCL-75

Optimal CLB-Transfection Pulse CELL 7 CELL 7 CELL 4 CELL 10 CELL 1 CELL 9 CELL 7 CELL 9 CELL 1 CELL 9 CELL 2 CELL 7 CELL 5 CELL 8 CELL 3 CELL 1 CELL 9 CELL 9 CELL 3 CELL 7 CELL 8 CELL 9 CELL 3 CELL 5 CELL 7 CELL 2 CELL 9 CELL 10 CELL 9 CELL 9 CELL 7 CELL 9 CELL 10 CELL 10

Transfection Efficiency (%) 80 60 75 80 41 56 55 59 35 70 45 98 91 82 67 25 52 45 65 53 34 30 30 55 32 39 65 60 37 56 95 38 80 75

Viability (%) 63 46 55 80 70 82 50 96 80 70 66 79 88 80 60 40 67 50 60 74 86 40 60 82 44 58 82 90 98 54 60 80 65 55

In case your cell line of interest is not listed in the CLB-Transfection Cell List, cell we recommend performing an optimization of the transfection conditions (please see Chapter 4.3).

CLB-Transfection Cell List 5.0

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6.0 Transformationo f Bacteria

The CLB-Transfection Device provides 4 preset pulses to optimize the electroporation conditions for transformation of different bacterial strains. Note: The CLB-Transfection Buffer and the certified cuvettes of the CLB-Transfection Kit are not suitable for bacterial transformation. Required materials: Sterile water for injection (transformation buffer) Standard 1 mm electroporation cuvettes LB or YT medium Plasmid DNA 1.5 l microcentrifuge tube One transformation sample contains: 20 l freshly thawed electrocompetent E. coli Up to 5 g plasmid DNA Note Electrocompetent E. coli can either be bought from different sources or self-prepared according to standard procedures. 1. 2. Thaw required aliquots of electrocompetent E. coli. Pre-warm an aliquot of culture medium containing supplements at room temperature in a 50 ml tube (200 l per sample). Mix 20 l of E. coli suspension with a maximum of 5 l plasmid DNA. Transfer a 20 l sample into a standard 1 mm cuvette. Make sure that the sample covers the bottom of the cuvette, avoid air bubbles while pipetting. Close the cuvette with the cap. No cooling of the cuvette is required. Insert the cuvette into the cuvette holder and rotate the carousel clockwise to the final position. Select the appropriate CLB-Transfection Pulse for bacterial transformation. Please initially try all 4 CLB-Transfection Pulses for bacterial transformation (BAC 1, BAC 2, BAC 3 and BAC 4) to determine the most appropriate one for all subsequent experiments. Apply the selected pulse by pressing the Start button. Only these specialized pulses for bacterial transformation will be efficient. 6. Take the cuvette out of the holder once the pulse is finished. 7. Add 100 200 l of the pre-warmed culture medium to the cuvette and transfer the sample into a 1.5 ml microcentrifuge tube and place it in a 37C heat block. 8. Press the Start button (5) to confirm the notification by the CLB-Transfection Device. 9. Incubate the diluted E. coli at 37C for expression of the resistance marker (e.g. for 30 minutes for ampicillin resistance and 60 minutes for tetracycline resistance). 10. Plate E. coli on plates with selective antibiotics according to standard procedures, e.g. in different dilutions or volumes to ensure separated single colony growth. 5.

3. 4.

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7.0 Troubleshooting
The following troubleshooting guide may be helpful if experiments using the CLB-Transfection Device do not work as expected. The listed comments are intended to help optimize experimental conditions. Should you have any questions regarding the CLB-Transfection Device or Procedure, please do not hesitate to contact Lonzas Scientific Support Team. Europe/World Phone +49-221-99199-400 Fax +49-221-99199-499 scientific.support.eu@lonza.com USA Phone 800-521-0390 (toll-free) Fax 301-845-8338 scientific.support@lonza.com

Problem Low survival rate

Possible error Cells are damaged by harvesting or through handling. Cells are stressed by culture conditions.

What happened? Avoid severe conditions during harvesting, especially centrifugation at higher speed and overexposure to trypsin. Pipette cells smoothly. Add 500 l pre-warmed medium to the cuvette before removal of the cells. Cells should be viable and in culture for a certain number of passages. Freshly thawed cells should not be used for transfection. Avoid excessive cell densities or cell confluencies, since this may negatively influence the viability of the cells after transfection. Centrifuge at lower speed (90xg). Mycoplasma infection might negatively influence experimental results. We recommend using the MycoAlert Mycoplasma Detection Kit (Lonza) to detect possible mycoplasma infections and MycoZap Mycoplasma Elimination Reagent (Lonza) to eradicate mycoplasma. We strongly recommend using cuvettes only once, because the electric pulses that are applied drastically reduce their quality and impair their physical integrity. Plasmid DNA used should be of high purity. We strongly recommend the use of high quality products for plasmid purification. Do not use procedures involving phenol or chloroform treatment.

Cells are stressed by centrifugation. Possible mycoplasma contamination.

Multiple use of cuvettes. Poor DNA quality.

Troubleshooting 7.0

16

Problem Low gene transfer

Possible error Plasmid amount is too low.

What happened? We recommend a plasmid amount between 1 5 g DNA per sample. If both gene transfer efficiency and cell mortality are low, the plasmid amount can be increased up to 10 g per sample. Increasing the DNA amount may lead to higher gene transfer efficiencies but at the same time may result in higher cell mortality. Gene transfer efficiency into many cell types is poor if the cells are too dense at the time of harvest. We recommend using 1 106 5 106 cells per sample. Mycoplasma infection might negatively influence experimental results. We recommend using the MycoAlert Mycoplasma Detection Kit (Lonza) to detect possible mycoplasma infections and MycoZap Mycoplasma Elimination Reagent (Lonza) to eradicate mycoplasma. Plasmid DNA used should be of high purity. We strongly recommend the use of high quality products for plasmid purification. Do not use procedures involving phenol or chloroform treatment.

High cell confluency/density. Too high or too low cell number in the cuvette. Possible mycoplasma contamination.

Poor DNA quality.

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8.0 CLB-Transfection Error Codes

The following guide indicates possible CLB-Transfection Error Codes and provides suggestions to solve the problem on your own. Should these suggestions not resolve the problem, please call Lonzas Scientific Support Team (Europe/World: +49 (0)221-99199-400; USA 800-521-0390). If the CLB-Transfection Device has to be returned for repair, please contact our Scientific Support Team for shipping and warranty instructions. Arc discharge correction Arcing is a complete or partial discharge circumventing the sample and is often accompanied by a flash and a noise. This problem is usually caused by imperfect cuvettes or cuvette filling. The CLB-Transfection Device is equipped with a hardware safety that immediately detects arc formation at its beginning to protect the cells from damage. After the arc interruption, the CLB-Transfection Device resumes pulse execution. Normally, the pulse can be completed successfully and only limited differences in transfection efficiency can be observed. When repeated arc discharges within one pulse occurs, it might be impossible for the CLB-Transfection Device to complete pulse execution. In this case (Error A) significant impacts on transfection efficiency might be detected. Discard the possibly damaged cuvette and its contents, reset the device by pressing the Start button (1), and repeat the experiment with a new cuvette. It is not necessary to switch off the CLB-Transfection Device.

Code Error A

Possible error Inappropriate or re-used cuvette, or inappropriate CLB-Transfection Buffer, or inappropriate volume in the cuvette or device possibly defective. Inappropriate cuvette, or inappropriate CLB-Transfection Buffer, or inappropriate volume or device possibly defective

What happened? Device possibly generated a suboptimal pulse due to inappropriate conditions regarding buffer, cuvette or volume.

Procedure Acknowledge the error message by pressing the Start button. Utilize sample if maximum efficiency is not essential. Otherwise, try a new sample and verify the volume in the cuvette is 100 l. Acknowledge the error message by pressing the Start button. Check If carousel is closed properly. Check the volume of CLB-Transfection Buffer. Try again with a new cuvette. Acknowledge the error message by pressing the Start button; switch off the device, wait for 5 seconds and switch on again. Try again with a new sample.

Error B

Pulse interrupted or no pulse generated. A substantial impairment on efficiency and viability has to be presumed.

Error C

Internal error or device possibly defective.

Pulse generated with unclear outcome or no pulse generated.

CLB-Transfection Error Codes 8.0

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9.0 Appendix
Technical data Power supply 100-110 VAC or 230 VAC 50 60 Hz self-regulating 50 VA/fuse T630mA L250V +15C to +40C, non-condensing 2000 m above sea level EN 61010-1 UL 61010-1 IP 20 2.7 kg 6 lb 30 23 11 cm 11.8 9.6 4.3 in The manufacturing year is encoded by the second and third digit of the serial number, e.g. serial number x09xxxxx was manufactured in 2009.

Power consumption Temperature range Altitude Safety class

Weight Dimensions (w d h) Manufacturing date

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Notes

Appendix 9.0

Contact Information
North America Customer Service: Scientific Support: scientific.support@lonza.com Online Ordering: Europe Customer Service: Cell Discovery Scientific Support: scientific.support.eu@lonza.com Molecular Biology, RTS & Media Scientific Support: scientific.support.eu@lonza.com Online Ordering: 800 638 8174 (toll free) 800 521 0390 (toll-free) www.lonza.com + 32 87 321 611 + 49 221 99199 400
Lonza Cologne AG 50829 Cologne, Germany For Research Use Only. Not for use in diagnostic procedures. Manufacturer and distributor information: The CLB-Transfection Device is manufactured by Lonza Cologne AG, Nattermannallee 1, 50829 Cologne, Germany and distributed in the U.S. by Lonza Walkersville, Inc. (8830 Biggs Ford Road, Walkersville, MD 21793). The CLB-Transfection Technology, comprising CLB-Transfection Process, CLB-Transfection Device and CLB-Transfection Buff er is covered by patent and/ or patent pending rights owned by Lonza Cologne AG. ATCC and the ATCC Catalog Marks are trademarks of ATCC used under License. Unless otherwise noted, all trademarks herein are marks of the Lonza Group or its affiliates.

+ 32 87 321 611 www.lonza.com

The use of this product, alone or in combination with materials and/or methods of others, may require a license from a third party. User shall be fully responsible for determining whether and from whom it requires such license and for obtaining such license. The information contained herein is believed to be correct and corresponds to the latest state of scientific and technical knowledge. However, no warranty is made, either expressed or implied, regarding its accuracy or the results to be obtained from the use of such information and no warranty is expressed or implied concerning the use of these products. The buyer assumes all risks of use and/or handling. No statement is intended or should be construed as a recommendation to infringe any existing patent. Copyright 2009, Lonza Cologne AG. All rights reserved. CD-MN007 05/09

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CD-MN007 05/09