Lonza Manual CLBTransfection

CLB-Transfection™ Manual

clb_transfection_manual_CS4.indd 2 18-6-09 20:05

Contents

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Contents

1.0 Introduction 3

2.0 Related Products 4

3.0 Operating Instructions 5

3.1 Restrictions 5

3.2 Maintenance 5

3.3 Safety Instructions – Please Read Carefully 6

3.4 Waste Disposal 7

3.5 CLB-Transfection™ Device Components 7

3.6 Setup Instructions 7

3.7 Instructions for Using the CLB-Transfection™ Device 83.7.1 Pulse Selection 83.7.2 Pulse Execution 8

3.8 Service Chipcard Slot 8

4.0 Transfection Procedure 9

4.1 Transfection of Suspension Cells Using the CLB-Transfection™ System 9

4.2 Transfection of Adherent Cells Using the CLB-Transfection™ System 10

4.3 Optimization of Transfection Conditions 11


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5.0 CLB-Transfection™ Cell List 12

6.0 Transformation of Bacteria 14

7.0 Troubleshooting 15

8.0 CLB-Transfection™ Error Codes 17

9.0 Appendix 18


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Introduction 1.0

1.0 Introduction

Lonza, the world’s 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 linesThe 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 choiceThe 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 substrateThe 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 over-expression studies, gene silencing approaches, protein expression, generation of stable clones and many other applications.

Components of the CLB-Transfection™ SystemThe 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 bacteriaIn addition to eukaryotic cell line transfections, the CLB-Transfection™ Device offers preset pulses for transformation of different bacterial strains.


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Cat. No. Product Size

AWC-1002 CLB-Transfection™ Device Guarantee - 2 year extension

VECA-1001 CLB-Transfection™ Kit 50 reactions

VK A-1001 Electroporation cuvettes for bacterial, 1 mm gap 50 cuvettes

LT07-218 MycoAlert® Mycoplasma Detection Kit 25 tests

LT07-121 ViaLight® Plus Cell Proliferation and Cytotoxicity BioAssay Kit 1000 tests

LT07-117 ToxiLight® Non-destructive Cytotoxicity BioAssay Kit 1000 tests

LT07-115 ApoGlow® Rapid Apoptosis Screening Kit 200 tests

VDF-1011 pmaxFP®-Green-C Vector 20 µg

VDF-1012 pmaxFP®-Green-N Vector 20 µg

VDF-1013 pmaxFP®-Green-PRL Vector 20 µg

VDF-1021 pmaxFP®-Yellow-C Vector 20 µg

VDF-1022 pmaxFP®-Yellow-N Vector 20 µg

VDF-1023 pmaxFP®-Yellow-PRL Vector 20 µg

VDF-1031 pmaxFP®-Red-C Vector 20 µg

VDF-1032 pmaxFP®-Red-N Vector 20 µg

VDF-1033 pmaxFP®-Red-PRL Vector 20 µg

VDC-1040 pmaxCloning™ Vector 20 µg

2.0 Related Products


Operating Instructions 3.0

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3.1 Restrictions

Medical use restrictionsThe CLB-Transfection™ System is intended for research and investigational use by professionals only. Please note that Lonza’s CLB-Transfection™ System is not intended to be used for diagnostic purposes or for testing or treatment in humans.

License statementLonza Cologne AG is holder of various patents, patent applications, copyrights and technical and scientific experience with respect to the CLB-Transfection™ Technology. Use of Lonza’s 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 Lonza’s 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 [email protected].

3.2 Maintenance

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.

3.0 Operating Instructions


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3.3 Safety Instructions – Please Read Carefully

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 user-serviceable 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.

— Use the device with Lonza’s certified CLB-Transfection™ Buffer and Lonza’s 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.

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.


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Operating Instructions 3.0

3.4 Waste Disposal

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

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

1 Power button2 Display 3 “Up” button 4 “Down” button 5 “Start” button6 Cuvette carousel7 Cuvette holder8 Service Chipcard Slot

Rear side of the CLB-Transfection™ Device

Figure 2

9 Power cord receptacle10 Spare fuse

3.6 Setup Instructions

1. Remove all packing material.2. 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 stand-by 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”.

910

581 74

2 3 6

Figure 1


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3.7 Instructions for Using the CLB-Transfection™ Device

3. 7.1 Pulse Selection

1. 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”).

2. 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.

3.7.2 Pulse Execution

1. 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.

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

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).

3.8 Service Chipcard Slot

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


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Electroporation Procedure 4.0

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 µg 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 4°C. 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 4°C.

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 supplier’s instructions

— Prewarm and equilibrate appropriate volume of culture medium to 37°C (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™ BufferAdd 1 ml CLB-Transfection™ Supplement to 4.5 ml CLB-Transfection™ Buffer and mix gently.

NotePlease 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 μl of CLB-Transfection™ Buffer plus 18 μl of CLB-Transfection™ Supplement to make 100 μl of total reaction volume.

One transfection sample contains: — 1 × 106 – 5 × 106 cells — 1 – 5 µg plasmid DNA (in 1 – 5 µl H2O or TE) or 30 – 300nM

siRNA (3 – 30 pmol/sample) — 100 µl CLB-Transfection™ Buffer

1. Prepare 12-well plates by filling the appropriate number of wells with 1 ml of supplemented culture medium and pre-incubate/equilibrate plates in a humidified 37°C/5% CO2

incubator. 2. Count an aliquot of the cells and determine cell density. 3. 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.

4. Resuspend the cell pellet carefully in 100 µl 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.

5. Combine 100 µl of cell suspension with 1 – 5 µg DNA or 30nM – 300nM siRNA (3 – 30pmol/sample) or other substrates.

4.0 Transfection Procedure


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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 µl 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 37°C/5% CO2 incubator until

analysis. Gene expression or down regulation, respectively, is often detectable after only 4 – 8 hours.

* If the cells grow in Dulbecco’s Modified Eagle Medium (DMEM) or Minimum

Essential Medium (MEM), we recommend using RPMI containing serum/

supplements.

4.2 Transfection of Adherent Cells Using the CLB-Transfection™ System

Preparation of the CLB-Transfection™ BufferAdd 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 4°C. Note date of addition on the vial.

NotePlease 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 μl of CLB-Transfection™ Buffer plus 18 μl of CLB-Transfection™ Supplement to make 100 μl of total reaction volume.

One transfection sample contains: — 1 × 106 – 5 × 106 cells — 1 – 5 µg plasmid DNA (in 1 – 5 µl H2O or TE) or 30 – 300nM

siRNA (3 – 30 pmol/sample) — 100 µl CLB-Transfection™ Buffer

1. 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 37°C/5% CO2 incubator.

2. Remove media from the cultured cells and wash cells once with PBS. Use at least same volume of PBS as culture media.

3. For harvesting, incubate the cells ~5 minutes at 37°C with 0.5 mg/ml Trypsin and 0.2 mg/ml EDTA.

4. Neutralize trypsinization reaction with supplemented culture medium or PBS/0.5% BSA once the majority of the cells (>90%) have been detached.

5. Count an aliquot of the cells and determine cell density. 6. 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.

7. Resuspend the cell pellet carefully in 100 µl 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.

8. Combine 100 µl of cell suspension with 1 – 5 µg DNA or 30nM – 300nM siRNA (3 – 30pmol/sample) or other substrates.

9. Transfer cell/DNA suspension into certified cuvette (sample must cover the bottom of the cuvette without air bubbles). Close the cuvette with the cap.

10. 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.

11. Take the cuvette out of the holder once the pulse is finished. 12. Immediately add 500 µl 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).


14. Incubate the cells in humidified 37°C/5% CO2 incubator until

analysis. Gene expression or down regulation, respectively, is often detectable after only 4 – 8 hours.

* If the cells grow in Dulbecco’s Modified Eagle Medium (DMEM) or Minimum

Essential Medium (MEM), we recommend using RPMI containing serum/

supplements.


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Electroporation Procedure 4.0

4.3 Optimization of Transfection Conditions

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)

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.

Well Pulse Transfection Efficiency Viability

1 CELL 1

2 CELL 2

3 CELL 3

4 CELL 4

5 CELL 5

6 CELL 6

7 CELL 7

8 CELL 8

9 CELL 9

10 CELL 10

11 No pulse

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


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The CLB-Transfection™ Cell List provides an overview about transfection conditions for a huge number of cell lines.

Cell Line Clone #Optimal

CLB-Transfection™ Pulse Transfection Efficiency (%) Viability (%)293 ATCC® CRL-1573 CELL 7 97 86

32D ATCC® CRL-11346 CELL 5 76 66

3T3-L1 ATCC® CL-173 CELL 8 50 70

A2058 ATCC® CRL-11147 CELL 9 75 48

A-431 ATCC® CRL-1555 CELL 10 54 62

A-549 ATCC® CCL-185 CELL 9 70 70

A7r5 ATCC® CRL-1444 CELL 9 57 60

AGS ATCC® CRL-1739 CELL 7 58 63

B16-F10 ATCC® CRL-6475 CELL 1 78 79

BA/F3 DSMZ ACC 300 CELL 9 90 61

BJ ATCC® CRL-2522 CELL 9 52 76

CCRF-CEM ATCC® CCL-119 CELL 9 59 50

CHO-K1 ATCC® CCL-61 CELL 9 84 91

COS-1 ATCC® CRL-1650 CELL 9 83 52

COS-7 ATCC® CRL-1651 CELL 9 98 95

FDC-P1 ATCC® CRL-12103 CELL 9 58 55

HCT 116 ATCC® CCL-247 CELL 5 78 60

HeLa ATCC® CCL-2 CELL 4 85 84

HeLa S3 ATCC® CCL-2.2 CELL 9 54 80

Hep G2 ATCC® HB-8065 CELL 8 50 95

HL-60 ATCC® CCL-240 CELL 8 61 74

HT-1080 ATCC® CCL-121 CELL 9 86 75

HT-29 ATCC® HTB-38 CELL 8 50 51

HuT 78 ATCC® TIB-161 CELL 10 53 64

HUV-EC-C ATCC® CRL-1730 CELL 10 48 61

IMR-90 ATCC® CCL-186 CELL 7 53 54

Jurkat (Clone E6-1) ATCC® TIB-152 CELL 9 52 75

K-562 ATCC® CCL-243 CELL 8 86 67

KG-1 ATCC® CCL-246 CELL 10 70 84

5.0 CLB-Transfection™ Cell List


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CLB-Transfection Cell List 5.0

Cell Line Clone #Optimal

CLB-Transfection™ Pulse Transfection Efficiency (%) Viability (%)

KG-1a ATCC® CCL-246.1 CELL 7 80 63

L428 DSMZ ACC 197 CELL 7 60 46

L6 ATCC® CRL-1458 CELL 4 75 55

LNCaP (Clone FGC) ATCC® CRL-1740 CELL 10 80 80

MCF7 ATCC® HTB-22 CELL 1 41 70

MDA-MB-453 ATCC® HTB-131 CELL 9 56 82

MDA-MB-468 ATCC® HTB-132 CELL 7 55 50

MDBK ATCC® CCL-22 CELL 9 59 96

MDCK (NBL-2) ATCC® CCL-34 CELL 1 35 80

MDCK II ECACC 00062107 CELL 9 70 70

MOLT-4 ATCC® CRL-1582 CELL 2 45 66

NCI-H1299 ATCC® CCL-246.1 CELL 7 98 79

Neuro-2a ATCC® CCL-131 CELL 5 91 88

NIH/3T3 ATCC® CRL-1658 CELL 8 82 80

NIH:OVCAR-3 ATCC® HTB-161 CELL 3 67 60

NK-92 ATCC® CRL-2407 CELL 1 25 40

NRK ATCC® CRL-6509 CELL 9 52 67

NS0 ECACC 85110503 CELL 9 45 50

P815 ATCC® TIB-64 CELL 3 65 60

PANC-1 ATCC® CRL-1469 CELL 7 53 74

PC-12 ATCC® CRL-1721 CELL 8 34 86

Raji ATCC® CCL-86 CELL 9 30 40

Ramos (RA 1) ATCC® CRL-1596 CELL 3 30 60

RAW 264.7 ATCC® TIB-71 CELL 5 55 82

RBL-2H3 ATCC® CRL-2256 CELL 7 32 44

S49 ATCC® TIB-36 CELL 2 39 58

SK-N-SH ATCC® HTB-11 CELL 9 65 82

SK-OV-3 ATCC® HTB-77 CELL 10 60 90

T/G HA-VSMC ATCC® CRL-1999 CELL 9 37 98

THP-1 ATCC® TIB-202 CELL 9 56 54

U-2 OS ATCC® HTB-96 CELL 7 95 60

U-937 ATCC® CRL-1593.2 CELL 9 38 80

Vero ATCC® CCL-81 CELL 10 80 65

WI-38 ATCC® CCL-75 CELL 10 75 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).


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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 µl microcentrifuge tube

One transformation sample contains: — 20 µl freshly thawed electrocompetent E. coli — Up to 5 µg plasmid DNA

NoteElectrocompetent E. coli can either be bought from different sources or self-prepared according to standard procedures.

1. Thaw required aliquots of electrocompetent E. coli. 2. Pre-warm an aliquot of culture medium containing

supplements at room temperature in a 50 ml tube (200 μl per sample).

3. Mix 20 μl of E. coli suspension with a maximum of 5 μl plasmid DNA.

4. Transfer a 20 μl 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.

6.0 Transformation o f Bacteria

5. 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 trans-formation (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 μl 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 37°C heat block.


9. Incubate the diluted E. coli at 37°C for expression of the resistance marker (e.g. for 30 minutes for ampicillin resist-ance 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.


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Troubleshooting 7.0

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 Lonza’s Scientific Support Team.

Europe/WorldPhone +49-221-99199-400Fax [email protected]

USA Phone 800-521-0390 (toll-free) Fax 301-845-8338 [email protected]

Problem Possible error What happened?

Low survival rate

Cells are damaged by harvesting

or through handling.

Avoid severe conditions during harvesting, especially centrifugation at higher

speed and overexposure to trypsin. Pipette cells smoothly. Add 500 µl

pre-warmed medium to the cuvette before removal of the cells.

Cells are stressed by culture

conditions.

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.

Cells are stressed by

centrifugation.

Centrifuge at lower speed (90xg).

Possible mycoplasma

contamination.

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.

Multiple use of cuvettes. We strongly recommend using cuvettes only once, because the electric pulses

that are applied drastically reduce their quality and impair their physical integrity.

Poor DNA quality. 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.


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Problem Possible error What happened?

Low gene transfer Plasmid amount is too low. We recommend a plasmid amount between 1 – 5 µg DNA per sample. If both

gene transfer efficiency and cell mortality are low, the plasmid amount can be

increased up to 10 µg per sample. Increasing the DNA amount may lead to higher

gene transfer efficiencies but at the same time may result in higher cell mortality.

High cell confluency/density. Gene transfer efficiency into many cell types is poor if the cells are too dense at

the time of harvest.

Too high or too low cell number in

the cuvette.

We recommend using 1 × 106 – 5 × 106 cells per sample.

Possible mycoplasma

contamination.

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.

Poor DNA quality. 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.


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CLB-Transfection™ Error Codes 8.0

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 Lonza’s 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 Possible error What happened? Procedure

Error A Inappropriate or re-used cuvette,

or inappropriate CLB-Transfection™

Buffer, or inappropriate volume

in the cuvette or device possibly

defective.

Device possibly generated

a suboptimal pulse due to

inappropriate conditions regarding

buffer, cuvette or volume.

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 µl.

Error B Inappropriate cuvette, or

inappropriate CLB-Transfection™

Buffer, or inappropriate volume or

device possibly defective

Pulse interrupted or no pulse

generated. A substantial

impairment on efficiency and

viability has to be presumed.

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.

Error C Internal error or device possibly

defective.

Pulse generated with unclear

outcome or no pulse generated.

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.


18

9.0 Appendix

Technical data

Power supply 100-110 VAC or 230 VAC

50 – 60 Hz

self-regulating

Power consumption 50 VA/fuse T630mA L250V

Temperature range +15°C to +40°C, non-condensing

Altitude ‹ 2000 m above sea level

Safety class EN 61010-1

UL 61010-1

IP 20

Weight 2.7 kg

6 lb

Dimensions

(w × d × h)

30 × 23 × 11 cm

11.8 × 9.6 × 4.3 in

Manufacturing date The manufacturing year is encoded by the second and third digit of the serial number, e.g. serial number x09xxxxx was

manufactured in 2009.


19

Appendix 9.0

Notes


Contact Information

North AmericaCustomer Service: 800 638 8174 (toll free)Scientifi c Support: 800 521 0390 (toll-free)scientifi [email protected] Ordering: www.lonza.com

EuropeCustomer Service: + 32 87 321 611Cell DiscoveryScientifi c Support: + 49 221 99199 400scientifi [email protected] Biology, RTS & MediaScientifi c Support: + 32 87 321 611 scientifi [email protected] Ordering: www.lonza.com

InternationalContact your local Lonza DistributorCustomer Service: + 1 301 898 7025, ext. 2322scientifi [email protected]

International Offi cesAustralia + 61 3 9550 0883Austria 0800 201 538 (toll free)Belgium + 32 87 321 611Brazil + 55 11 2069 8800Denmark + 45 43 56 74 00France 0800 91 19 81 (toll free)Germany 0800 182 52 87 (toll free)India + 91 22 4342 4000Italy + 39 0363 45710Japan + 81 3 5566 0612Poland + 48 22 833 87 45Singapore + 65 6 4914214Spain + 34 902 531 366Sweden 020 140 4410 (toll free)Switzerland 0800 83 86 20 (toll free)The Netherlands 0800 022 4525 (toll free)United Kingdom + 44 118 979 5234

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 manu-factured 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 affi liates.

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 scientifi c 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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Lonza Manual CLBTransfection

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