The Eppendorf Approach for Improved Workflows

Transcription

1 (BN 42) JANUARY 2015 PAGE 3 Abstract Multi-well plates are common tools in the analysis of cellular responses using cell-based assays. The so-called edge effect can lead to highly inconsistent results in the outer wells. Therefore often only 60 out of 96 wells are used for analysis which means a capacity decrease of 38 % per plate. With the Eppendorf 96-Well Cell Culture Plate the edge effect can be reduced to a minimum. This application note compares the performance of Eppendorf 96-Well Cell Culture Plates with 96-well cell culture plates from different manufacturers concerning well-to-well variability in evaporation.* Introduction Cell-based assays are of increasing importance in almost all fields of biological science. The significance and value of the obtained data relies on the consistency of the performed assays. There are many sources of assay inconsistency in multi-well plates. Some of these aspects can be avoided by optimizing routine procedures in the laboratory. Possible measures include the implementation of adequate mixing and dissociation of the cells, the prevention of air bubble formation during cell seeding and the determination of optimal growth conditions concerning media A JESSICA WAGENER, EPPENDORF AG, HAMBURG, GERMANY CHRISTELLE PLENNEVAUX, EPPENDORF APPLICATION TECHNOLOGIES, NAMUR, BELGIUM formulation and seeding density. However, the edge effect can rarely be ad- very carefully because some cell types room temperature must be considered dressed by these precautions. are particularly sensitive to temperatures below 37 C. As well-to-well variability mainly affects the peripheral wells of a plate this phenomenon is called the edge effect. The cubation instruments like humidity- and For applications requiring long-term in- causes of the edge effect are complex. temperature-chambers are available. Their purpose is the creation of a constant micro-environment by reducing Temperature differences across the plate and evaporation effects in the edge wells evaporation to a minimum and achieving almost consistent humidity and during incubation have been described as two possible influencing factors. It temperature levels. is assumed that evaporation in the outer wells may lead to an accumulation of Probably the most common method to medium components (e.g. salts), thereby affecting cell metabolism. Varying seeding cells in the peripheral wells. circumvent the edge effect is to avoid cell responses may also be the result of Not using the outer wells of a 96-well temperature gradients. Both factors plate, however, reduces the number of evaporation and temperature gradient wells for analysis to 60 and results in a may lead to higher variances between 38 % lower throughput per plate. This individual assays, thus resulting in unreliable data. requires higher investments for con- method may avoid the edge effect but sumables, incubator space, and time. There are different methods to prevent the edge effect. One approach to minimize the impact of a temperature gra- Material and methods Preparation of plates dient is the pre-incubation of the plate with freshly seeded cells at room temperature which results in a more even plates the Eppendorf Cell Culture Plate For evaluating evaporation in 96-well cell distribution and adhesion pattern. as well as three plates from other manufacturers (A, B, and C) were tested (n=2). However, the edge effect does not only affect consistent cell growth. Even the The Eppendorf Plate has a surrounding most homogeneous monolayer may moat that can be filled with liquid (e.g. lead to unreliable results. Depending sterile water or PBS), thus insulating the on the cell type used, pre-incubation at edge wells. Fig. 1: The Eppendorf 96-Well Cell Culture Plate, filling options. A) Outer moat filled with liquid to insulate specifically the edge wells B) Insulation of all 96 wells by filling the complete inter-well space Your local distributor: Eppendorf AG Hamburg Germany eppendorf@eppendorf.com B No The Eppendorf Approach for Improved Workflows > Fast and Precise Microplate Processing > Finest Products for Food and Beverage Labs > Tradition Continued: New Thermomixers Application Notes Eppendorf 96-Well Cell Culture Plates: Minimizing the Edge Effect in Cell-Based Assays PCR Optimization and Highly Flexible Operation on the Mastercycler nexus GX2 etc. Eppendorf 96-Well Cell Culture Plates: a Simple Method of Minimizing the Edge Effect in Cell-Based Assays

2 2 EDITORIAL DEAR READERS Imprint Editorial team Berrit Hoff (Editor-in-Chief), Axel Jahns, Jochen Müller-Ibeler, Natascha Weiß Publisher Eppendorf AG, Barkhausenweg 1, Hamburg, Germany Telephone: (+49) Fax: (+49) Internet: Dear Readers, we are pleased to present you with more smart solutions that allow convenient, safe, and dependable processes in the laboratory in this edition of BioNews. Efficient, durable instruments with matching consumables and custom accessories facilitate your daily routine with common workflows such as cell biology (p. 4 5) as well as in specialized laboratories, e. g. food technology (p ). Eppendorf products are always fine-tuned to each other, so you may concentrate on what really matters your research! Two of the most frequently used laboratory vessel formats are now also available from Eppendorf. The new Eppendorf Conical Tubes 15 ml and 50 ml with screw cap are versatile and convincing all the way; you are invited to read all about the new tubes on p. 9 as well as in the Application Notes on pages 7 8. We welcome all readers articles for this publication. However, no responsibility is accepted for unsolicited manuscripts. Important note The new products described may be launched at different times in various countries. Please contact your local Eppendorf organization or distributor for details. Technical specifications subject to change. Errors and omissions excepted. All rights reserved, including graphics and images. Copyright Eppendorf AG, January Carbon neutrally printed in Germany. For 50 years, Eppendorf has been setting the standard in the areas of mixing and temperature control of small volumes. Our latest models, the Eppendorf ThermoMixer F0.5 and F2.0, respectively, are the perfect choice for routine applications in 0.5 ml and 2.0 ml tubes (p. 12). Our new column Close-up focusses on product details which are meaningful for extraordinary efficiency and high-quality results. Learn more about the certain Eppendorf-Extra! First up is the Eppendorf µcuvette G1.0 (p. 11). In-depth Application Notes as well as the popular competition with great prizes will round off this new edition of Eppendorf BioNews. Enjoy a good read! Your Eppendorf BioNews editorial team PS: Did you know that Eppendorf BioNews has been around for 22 years? We would like to find out from you what you like or what we could improve. More information about our survey is available in the box on p. 9.

3 (BN 42) JANUARY 2015 PAGE 3 JESSICA WAGENER, EPPENDORF AG, HAMBURG, GERMANY CHRISTELLE PLENNEVAUX, EPPENDORF APPLICATION TECHNOLOGIES, NAMUR, BELGIUM Fig. 1: The Eppendorf 96-Well Cell Culture Plate, filling options. A) Outer moat filled with liquid to insulate specifically the edge wells B) Insulation of all 96 wells by filling the complete inter-well space Your local distributor: Eppendorf AG Hamburg Germany eppendorf@eppendorf.com CONTENTS IN THE SPOTLIGHT Improve Your Laboratory Workflows: Take the Eppendorf Approach! 4 5 STRAIGHT FROM THE LAB Much More Than Bioprocess Control 6 7 Finest Products for Food and Beverage Labs 10 INNOVATION Fast and Precise Microplate Processing 8 No Compromises from 0.5 ml to 50 ml! 9 CLOSE-UP Eppendorf μcuvette G NEWS / TIPS NEW: Scan Your eppoints Codes! 5 ULT Freezer Performance Plans 7 Get New Features for Your Older epmotion And what do YOU think of us? BioNews Online Survey 9 Tradition Continued: New Thermomixers 12 User Test of TransferMan 4r 12 Eppendorf Research Prizes: Winners SERVICE Prize Competition 14 Reply Fax / Readers Service 15 Eppendorf 96-Well Cell Culture Plates: a Simple Method of Minimizing the Edge Effect in Cell-Based Assays Abstract formulation and seeding density. However, the edge effect can rarely be ad- very carefully because some cell types room temperature must be considered Multi-well plates are common tools in dressed by these precautions. are particularly sensitive to temperatures below 37 C. the analysis of cellular responses using cell-based assays. The so-called edge As well-to-well variability mainly affects effect can lead to highly inconsistent the peripheral wells of a plate this phenomenon is called the edge effect. The cubation instruments like humidity- and For applications requiring long-term in- results in the outer wells. Therefore often only 60 out of 96 wells are used for causes of the edge effect are complex. temperature-chambers are available. analysis which means a capacity decrease Their purpose is the creation of a constant micro-environment by reducing Temperature differences across the plate of 38 % per plate. With the Eppendorf and evaporation effects in the edge wells 96-Well Cell Culture Plate the edge evaporation to a minimum and achieving almost consistent humidity and during incubation have been described effect can be reduced to a minimum. as two possible influencing factors. It This application note compares the temperature levels. is assumed that evaporation in the outer performance of Eppendorf 96-Well Cell wells may lead to an accumulation of Probably the most common method to Culture Plates with 96-well cell culture medium components (e.g. salts), thereby affecting cell metabolism. Varying seeding cells in the peripheral wells. circumvent the edge effect is to avoid plates from different manufacturers concerning well-to-well variability in cell responses may also be the result of Not using the outer wells of a 96-well evaporation.* temperature gradients. Both factors plate, however, reduces the number of Introduction evaporation and temperature gradient wells for analysis to 60 and results in a may lead to higher variances between 38 % lower throughput per plate. This Cell-based assays are of increasing importance in almost all fields of biological individual assays, thus resulting in unreliable data. requires higher investments for con- method may avoid the edge effect but science. The significance and value of sumables, incubator space, and time. the obtained data relies on the consistency of the performed assays. There the edge effect. One approach to mini- Material and methods There are different methods to prevent are many sources of assay inconsistency in multi-well plates. Some of these dient is the pre-incubation of the plate mize the impact of a temperature gra- Preparation of plates aspects can be avoided by optimizing with freshly seeded cells at room temperature which results in a more even plates the Eppendorf Cell Culture Plate For evaluating evaporation in 96-well routine procedures in the laboratory. Possible measures include the implementation of adequate mixing and dis- However, the edge effect does not only facturers (A, B, and C) were tested (n=2). cell distribution and adhesion pattern. as well as three plates from other manusociation of the cells, the prevention of affect consistent cell growth. Even the The Eppendorf Plate has a surrounding air bubble formation during cell seeding most homogeneous monolayer may moat that can be filled with liquid (e.g. and the determination of optimal lead to unreliable results. Depending sterile water or PBS), thus insulating the growth conditions concerning media on the cell type used, pre-incubation at edge wells. A B BIN LI, STACEY WILLARD, MA SHA High Cell Density Fermentation of Escherichia coli Using the New Brunswick BioFlo 115 JESSICA WAGENER, CHRISTELLE PLENNEVAUX Eppendorf 96-Well Cell Culture Plates: a Simple Method of Minimizing the Edge Effect in Cell-Based Assays NILS GERKE, ANDREA HELLBERG Straightforward PCR Optimization and Highly Flexible Operation on the Dual Block Thermocycler Mastercycler nexus GX RAFAL GRZESKOWIAK, KATJA KAROW Performance Comparison of Eppendorf Conical Tubes: Cap Tightness, Centrifugation Stability, and Leachables Levels 7 8

4 4 IN THE SPOTLIGHT IMPROVE YOUR LABORATORY WORKFLOWS: TAKE THE EPPENDORF APPROACH! TANJA MUSIOL & NATASCHA WEISS, EPPENDORF AG Improve Your Laboratory Workflows: Take the Eppendorf Approach! For nearly 70 years, Eppendorf s innovative technologies and premium products have contributed to improving work processes in liquid, cell, and sample handling in laboratories and research facilities all over the world. Our portfolio comprises highly efficient, durable instruments, matching consumables, and tailor-made accessories designed to support a great variety of workflows and facilitate our users daily lab routine. Additionally, dedicated epservices programs ensure that our customers instruments are always operating at peak performance. Fig. 1: Eppendorf s smart solutions for convenient, safe, and dependable processes in the cell biology lab. Your guarantee for reliable and reproducible results. Eppendorf has never been resting on its laurels. We have always been committed to fulfill the highest expectations of our customers. A long-standing, close relationship to the scientific community has given us a deep understanding of our users lab routines. Our expertise and creativity drive us to keep investing in a continually growing portfolio of premium products and services, with unwavering focus on top quality. Eppendorf users can always depend on new, future-oriented product groups, improved successor products, and state-of-the art applications. That s the Eppendorf approach! Now, take a closer look at the workflows for which Eppendorf can offer you a wide range of intelligent solutions. Cell biology (eukaryotic cells) Cell biology is a branch of biology that studies cells the organelles they contain, their function, their physiological properties, their life cycle, the interactions with their environment, etc. Cell biology basic research can be divided into several subfields, e.g. the study of cell metabolism, the study of cell genetics and the underlying regulatory mechanisms, the study of cell compartment structures, the study of cell cycle, division, and death, and the study of cell communication and signaling. Research in cell biology overlaps to a great extent other areas of biology and chemistry, particularly genetics, biochemistry and molecular biology. Eppendorf offers premium products for all major working steps within cell biology:

5 IMPROVE YOUR LABORATORY WORKFLOWS: TAKE THE EPPENDORF APPROACH! IN THE SPOTLIGHT 5 cell manipulation, cell seeding, cell cultivation, harvesting and preparation, analysis and detection, and storage (Fig. 1). Molecular biology The goal of molecular biology is to analyze and understand the molecular basis of biological processes. It concerns itself with the function of DNA/RNA sequences and the interaction between DNA, RNA, and proteins. Furthermore, this field overlaps to a great extent with other areas of biology and chemistry, particularly biochemistry and genetics. One of the most important molecular biology methods is the polymerase chain reaction (PCR). It is used to exponentially amplify a specific segment of DNA for various downstream applications like DNA cloning (genetic engineering), gene expression analysis (RT-PCR, RT-qPCR), genetic fingerprinting (paternity testing, forensics), sequencing (Next Generation Sequencing), and others more. Microbiology (microorganisms) Microbiology is the study of microorganisms, a large and diverse group of organisms that exist as single cells or cell clusters. It is a broad biological discipline which includes basic research as well as applied science. The identification of microorganisms is part of medical microbiology as well as quality testing e.g. in pharmaceutical and food industry. Examples for the usage of microbes in industrial processes include fermentation (e.g. production of food and beverages, drugs, vaccines, biofuels, enzymes) and wastewater treatment. Biochemistry Classical protein biochemistry explores the biological function of a protein, its amino acid composition, its structure and binding partners, the subcellular localization of the protein, and finally its physiological role in the organism. Besides applications in research, biochemical methods are used in the food industries for the detection of allergens and pathogens, in drug discovery for the identification and validation of target molecules, and for the analysis of transgenic organisms. An additional important field is biotechnological protein production (e.g. enzymes, antibodies). Next Generation Sequencing (NGS) DNA sequencing is a method to decipher the base sequence in nucleic acids. The elucidation of the DNA sequence is essential for the understanding of virtually all biological processes, e.g. human disease biology, inheritance, immunology, oncology, and cellular biology. First generation sequencing devices use the capillary electrophoresis-based Sanger sequencing technique. The invention of the Next Generation Sequencing technology has strongly improved the weaknesses of the 1st generation sequencing technology such as low throughput, scalability, speed, and resolution. NGS allows massive parallel processing which reduces the costs and increases the speed of DNA sequencing. Bioprocessing R&D (microorganisms and cells) Research and process development are key elements in the creation of improved, more rapid, and lower-cost methods for producing bio-based products. Working with small volumes saves valuable resources while industry-standard bioprocess systems deliver the precision required to operate under production-like conditions. Advanced software solutions feature realtime process control as well as comprehensive data and information management. The Quality by Design (QbD) approach and statistic tools such as Design of Experiments (DoE) are effective means for a faster development. Single-use bioreactors further improve turn-around times and simplify validation. Being in process with Eppendorf! You want to know more? Our new website has been specially designed to illustrate Eppendorf s product range for select workflows! Here you will also find detailed application notes. Tip NEW: Scan Your eppoints Codes! Entering your eppoints codes is now much easier! Many new eppoints stickers carry a QR code which allows you to scan your eppoints codes using the camera of your mobile device, for example your smartphone or tablet PC. It s easy: 1. Open your preferred QR code scanner app on your mobile device. 2. Scan the QR code on the eppoints sticker by using the camera of your mobile device. 3. Log in with your eppoints user data (if you haven t done so already) or open an eppoints account at 4. Choose your product from the list. 5. Click Go. 6. You can easily scan another code by repeating steps 1, 2, 4, and 5. If you prefer to enter the code without scanning it or if your box contains a sticker without a QR code, you can still enter the code easily by hand. All older eppoints codes remain valid! eppoints is Eppendorf s customer rewards program. You can find eppoints stickers in almost every box of Eppendorf consumables. Try it! For more information scan the QR code below or visit the eppoints mobile website: m.eppoints.com

6 6 STRAIGHT FROM THE LAB MUCH MORE THAN BIOPROCESS CONTROL CLAUDIA M. HÜTHER-FRANKEN, EPPENDORF AG, BIOPROCESS CENTER EUROPE Much More Than Bioprocess Control For decades, bioreactor processing, recipe management, process and product analysis, data generation and storage were poorly interconnected and required time-intensive manual work. With the Eppendorf DASware bioprocess software interconnectivity is combined with sophisticated bioprocess information management. DASware enables streamlined process development at benchtop scale, in accordance with Quality by Design (QbD) standards. The OPC network protocol allows for interconnectivity between the bioreactor system and the analyzer, including the possibility of direct feedback from the bioreactor system according to online measured analytical data. This facilitates feedback control loops for e.g. nutrients, biomass or product concentrations. Online calculations as well as event- and data-driven decisions are supported. The unique bidirectional OPC communication available for supporting devices enables sampling on demand. Design of Experiments (DoE) DASware design automatically compiles DoE information from third-party DoE tools into recipes and feedback response information into DoE and multivariate analysis and reporting tools. Process complexity and rising cost pressures make today s process development a special challenge. Achieving faster timeto-market for new and innovative biotechnological products requires optimization of every element of the entire development workflow. Eppendorf DASware was designed as a suite of smart and flexible software solutions to accelerate bioprocess development. It can be used with all Eppendorf DASGIP and New Brunswick benchtop bioreactor lines as well as with third-party controllers. Analyzer integration DASware analyze was designed for seamless integration of laboratory devices into the bioreactor system. A broad range of analyzers like nutrient analyzers and cell counters, autosamplers, biomass monitors, HPLCs, and mass spectrometers can be integrated. DASware design comes with a full factorial DoE builder. Alternatively, a large variety of DoE designs for screening, process development and optimization can be automatically imported from most powerful third-party DoE tools. Parallel recipes incorporating the DoE factor variations (i.e. ph, DO, T setpoints or feedrates) are automatically populated. Following the user-friendly Point-Click- Grow concept they can be carried out on a set of bio reactors with a single mouse-click. DoE response information is collected and prepared for an automated export.

7 MUCH MORE THAN BIOPROCESS CONTROL STRAIGHT FROM THE LAB 7 News ULT Freezer Performance Plans Whether you use a space-saving Innova freezer, a cost-saving instrument from our Premium series, or a highly efficient model from our HEF and Green G product range, your demands on an ultra-low temperature freezer are clearcut! It must preserve and protect your extremely valuable biological samples reliably and over many years. The unique DASGIP iapp supports access from iphone, ipod touch, and ipad. Comprehensive data management, remote control, and company wide data access With DASware discover both configurable and retrievable critical process parameters can be added to process runs either online or retrospectively. DASware discover enables near real-time retrieval of runtime information by intuitive Microsoft Excel style queries. A report generator provides recipe information, process information as well as event reporting. Utilizing the integral chart creator tool users can simultaneously compare process information from either current or historical runs. DASware access provides freedom and flexibility in bioprocess management. Each bioreactor on-site is accessible remotely by one or more remote clients. Wi-Fi, Intranet, VPN, and 3G connections can be used to provide web-based access with almost any browser to one or more bioreactor systems via PC, notebook, or netbook. The unique DASGIP iapp supports access from iphone, ipod touch and ipad, optionally with webcam support. DASware connect was designed to integrate Eppendorf and third-party benchtop bioreactor controllers into process control systems and legacy corporate historians. It facilitates company-wide access to all relevant bioprocess data like set-points, process values, feedprofiles, calibration and controller parameters as well as events and alarms. Gateway to third-party bioreactor controllers DASware migrate was designed to facilitate integration of different bioreactor control units and systems. It enables access to advanced control features and powerful Microsoft Excel reporting as well as the suite of DASware solutions such as integration of third-party lab de vices, support of DoE, comprehensive data- and information management, and interconnection to process control systems and corporate historians. DASware migrate easily integrates the Eppendorf exhaust analyzer DASGIP GA4, biomass monitor OD4, precision multi pumps MP8 and gas mixing stations MX4/4 into third-party bioreactor controllers. All-in-one solution DASware users in biotech, pharma, and chemical industries benefit from a professionally integrated, open, and extendable platform that enables Process Analytical Technology (PAT), feedforward and feedback loops as well as DoE and multivariate analysis. Using DASware facilitates to merge independent poorly automated workflows in the laboratory into a QbDdriven, integrated, and well documented development process. This saves development time and reduces time-tomarket. Brochure We Know Bioprocessing Ref. no. 274 Reliable refrigeration and optimized instrument conformity with programmed storage temperatures will assist you to store your frozen samples safely. We provide service programs to ensure your freezer is continuously working within the manufacturer specifications, giving you peace of mind. The Eppendorf ULT Freezer Performance Plans feature: > > A choice of preventive maintenance programs designed to check and insure continuous stable freezer performance > > Validation and adjustment of operating parameters in accordance to Eppendorf specifications > > Installation Qualification certification > > Operational Qualification certification > > Full service documentation Your benefits: > > Minimized risk of system failure > > Long lifetime of your instrument > > Confirmation of instrument performance within manufacturer s specifications For more information please visit or local websites.* *Performance Plans (also for other Eppendorf products) are available in selected countries.

8 8 INNOVATION FAST AND PRECISE MICROPLATE PROCESSING CARSTEN BUHLMANN, EPPENDORF AG Fast and Precise Microplate Processing Scientists facing the sometimes pesky task of repetitive pipetting into 96- or 384-well microplates or deepwell plates will be pleased: Eppendorf s new semi-automated electronic pipette epmotion 96 provides a time-saving solution for tasks like cell seeding, media change, or compound/reagent addition. Tip Get New Features for Your Trusted epmotion 5075 Handling: very fast, very easy Compared to manual multi-channel pipettes the handling is much easier and faster with high precision, accuracy, and reproducibility. The epmotion 96 dispenses into all 96 wells in a synchronous fashion, enabling for example a simultaneous start or stop of a biological assay in all wells of the plate. Reduced RSI risk With the epmotion 96 repetitive pipetting becomes more comfortable and efficient. The epmotion 96 furthermore greatly reduces the risk of Repetitive Strain Injury (RSI) that is often caused by manual pipetting. Thus, it fulfills the criteria of the Eppendorf PhysioCare Concept. Large volume range The epmotion 96 has a large volume range between 0.5 µl and 300 µl using only one head or system. There is no need to switch pipette heads or employ a second device to achieve all volumes. Working modes include Multidispense, Pipette + Mix, Manual Pipette, Dilute + Mix, Multiaspirate, with various pipetting settings. Easy, intuitive use is ensured by the proven Eppendorf software concept and a large touch screen control. With the new epmotion 96 users get the exact results they need for a wide range of applications and methods. Due to its small footprint it is perfectly suited for use in busy laboratories or under a laminar flow hood. If you want to learn more please request the product brochure or visit With the new epmotion 96 you can fill entire 96-well plates at once, which greatly enhances your productivity and efficiency. An adapter is provided for 384-well plates. The latest epmotion models (introduced in October 2013) offer more worktable positions and a higher speed of the robotic arm than previous versions. But that s not all! New features like, for example, re-use of tips and multi-step liquid transfers have expanded their performance and efficiency even more. The good news is that these new great features are now also available for older models! The epmotion 5075 Software & Hardware Upgrade Set has been specially developed to raise old style epmotion 5075 PC or control panel versions to the same performance level. Upgraded systems will have no functional difference to new versions and will benefit from all additional features, speed, and deck positions. Because the new MultiCon touch PC is operated by Windows 7 the set is also a great solution for users of 5075 PC versions looking for an alternative to Windows XP that is no longer supported by Microsoft. Upgrade now! To learn more about the new epmotion systems and features order the epmotion Family Brochure or visit If you are interested in upgrading your old epmotion 5075 with the new sets of features please contact your local Eppendorf sales representative. epmotion 96 Ref. no. 275 epmotion Family Brochure Ref. no. 254

9 (BN 42) JANUARY 2015 PAGE 1 High Cell Density Fermentation of Escherichia coli Using the New Brunswick BioFlo 115 BIN LI, STACEY WILLARD, AND MA SHA, EPPENDORF, INC., ENFIELD, CT, USA Abstract This application note presents a successful example of a high density fermentation of Escherichia coli (E. coli) using the New Brunswick BioFlo 115 benchtop, autoclavable fermentor. The highest optical density (OD 600 ) achieved in this study was 140 at 11 hours (h) without optimized fermentation medium and conditions. Introduction E. coli is a Gram-negative bacterium that has had a long history in the world of laboratory and industrial processes due to its ease of manipulation and well understood genome. It is widely cultivated under aerobic conditions. High cell density fermentation of E. coli is a powerful technique for the production of recombinant proteins. In this application note, E. coli cultivation achieved a high OD 600 value of 140 at 11 h using fed-batch fermentation with the New Brunswick BioFlo 115 benchtop fermentor (Fig. 1). Materials and methods Equipment The E. coli K12 strain (ATCC, ) was grown in a 2 L working volume New Brunswick BioFlo 115 heat-blanketed glass vessel (Eppendorf). Glucose concentrations were measured using a Cedex Bio Analyzer (Roche ). The OD 600 was measured with an Epoch Microplate Spectrophotometer (BioTek ) using the optional cuvette attachment. Medium The initial fermentation medium was prepared as follows: 150 ml 10 X phosphate/citric acid buffer [133 g/l KH 2 PO 4, 40 g/l (NH 4 ) 2 PO 4, 17 g/l citric acid] and 1.35 L deionized (DI) water were added to the vessel before sterilization at 121 C for 20 min. After the solution was cooled to room temperature, the following sterile components were added to make the complete fermentation medium: 15 ml of 240 g/l MgSO 4, 0.34 ml of 20 g/l thiamine, 15 ml of 100 X trace element solution, and 22 ml of 70 % glucose solution. The 100 X trace element solution contained: 10 g/l iron (III) citrate, 0.25 g/l CoCl 2 6 H 2 O, 1.5 g/l MnCl 2 4 H 2 O, 0.15 g/l CuCl 2 6 H 2 O, 0.3 g/l H 3 BO 3, 0.25 g/l Na 2 MoO 4 2 H 2 O, 1.3 g/l zinc acetate 2 H 2 O, 0.84 g/l EDTA. The ph was adjusted to 6.8 using 25 % (v/v) NH 4 OH or 6 N HCl. An additional concentrated feeding medium was prepared separately in a 1 L glass bottle. 45 ml of 240 g/l MgSO 4, 1.66 ml of 20 g/l thiamine solution, 15 ml of 100 X trace element solution, and 70 % glucose solution were added to a final volume of 500 ml. Inoculum and fermentation The inoculum was grown in Terrific Broth (TB) medium, prepared as described previously [1]. Two 500 ml baffled shake flasks (VWR, ) each containing 100 ml of TB medium were inoculated from a frozen vial of E. coli and incubated at 30 C, 200 rpm overnight in a New Brunswick Innova 40 benchtop incubator shaker (Eppendorf). After the overnight culture, the OD 600 value was ~9. The vessel was inoculated with 75 ml of inoculum (5 % of the initial working volume). Initial fermentation temperature was set to 30 C. Antifoam 204 (Sigma- Aldrich, A6426) was added only when needed, since it may reduce the oxygen transfer rate (OTR) and possibly lower the final cell density. In this experiment, ~100 µl of 5 g/l antifoam 204 was added at the beginning of the run to prevent foaming and ~4 ml was added between 8 11 h of fermentation, as foam accumulation warranted. Pump 3 was assigned as the feeding pump (max. speed is 24 ml/min with 4.78 mm [0.188 in] inner diameter tubing). Fig. 1: The New Brunswick BioFlo 115 benchtop fermentor with water-jacketed (left) and heat-blanketed (right) vessels The feeding strategy included increasing or decreasing the feeding pump speed accordingly, based on the glucose concentration. To achieve high cell density, the target glucose concentration was 2 g/l. Your local distributor: Eppendorf AG Hamburg Germany eppendorf@eppendorf.com

10 PAGE 2 (BN 42) JANUARY 2015 High Cell Density Fermentation of Escherichia coli Using the New Brunswick BioFlo 115 A [Glucose] (g/l) [Glucose] OD Time (h) OD 600 B ln (OD 600) y = 0.58 x 1.01 R 2 = Time (h) Fig. 2: Fermentation growth curve and growth rate calculation. A) The OD 600 and glucose concentration over the course of the 11 h fermentation B) The growth curve plotted on a log scale; a linear trend line was applied in Microsoft Excel, the slope of which is equivalent to the specific growth rate, µ (h 1 ) Cell growth and glucose concentration were monitored offline using 5 ml samples taken according to the following schedule. For OD 600 readings, samples were taken every hour and diluted appropriately for accurate measurement. For the determination of glucose concentration, samples were taken every hour before the initiation of feeding, and then every ~30 min after the feeding began. The specific growth rate (µ) was calculated from the fitted OD 600 value in Microsoft Excel. ph calibration and control ph calibration was done outside the vessel using a two-point calibration method and standard buffers. Buffer ph 7.0 was used to set ZERO and ph 4.0 for the SPAN. The ph sensor was calibrated prior to autoclaving the vessel. The ph was automatically maintained at 6.8 by adding 6 N HCl via pump 1 (assigned as acid ) to lower the ph and adding 25 % (v/v) NH 4 OH via pump 2 (assigned as base ) to raise the ph. The deadband for ph control was set to Dissolved oxygen (DO) sensor calibration and gassing control DO sensor calibration was performed using a standard two-point calibration method: 0 % (set ZERO ) was obtained by disconnecting the sensor from the cabinet and allowing the raw value to stabilize; 100 % (set SPAN ) was obtained by running 1,200 rpm agitation and 3 SLPM air flow until the DO value stabilized at maximum. The New Brunswick BioFlo 115 Reactor Process Controller (RPC) software offers a selection of automatic gassing control cascades that are dependent upon the configuration of the unit. The New Brunswick BioFlo 115 used in this study included the automatic gas mix option and one TMFC with a flow range of 0 20 SLPM. Operating in fermentation mode, the automatic DO cascade Agit/GasFlo/O2 was selected with a DO setpoint of 30 %. Results and discussion Samples were taken periodically to monitor the cell growth (OD 600 value) and glucose concentration. Feeding was initiated when the glucose concentration dropped below 2 g/l, which occurred at 5 h of cultivation. After starting the feed, the pump rate was adjusted according to the current glucose concentration with the end goal of keeping it at or below 2 g/l. As shown in Fig. 2A, within 11 h, the OD 600 value reached 140. The growth curve was also plotted on a log scale to calculate the specific growth rate (µ = 0.58 h 1, Fig. 2B). Conclusion High density E. coli growth in the New Brunswick BioFlo 115 was achieved using a fed-batch fermentation method. An optical density of 140 was reached at 11 h. Although efforts were made to maintain a glucose concentration below 2 g/l, the fermentation was not optimized for medium, growth conditions, or any product yield. Literature [1] Terrific Broth. Cold Spring Harbor Protocols 2006; 2006(1):pdb.rec8620. Readers service Brochure We Know Bioprocessing Ref. no. 274 Your local distributor: Eppendorf AG Hamburg Germany eppendorf@eppendorf.com

11 (BN 42) JANUARY 2015 PAGE 3 Eppendorf 96-Well Cell Culture Plates: a Simple Method of Minimizing the Edge Effect in Cell-Based Assays JESSICA WAGENER, EPPENDORF AG, HAMBURG, GERMANY CHRISTELLE PLENNEVAUX, EPPENDORF APPLICATION TECHNOLOGIES, NAMUR, BELGIUM Abstract Multi-well plates are common tools in the analysis of cellular responses using cell-based assays. The so-called edge effect can lead to highly inconsistent results in the outer wells. Therefore often only 60 out of 96 wells are used for analysis which means a capacity decrease of 38 % per plate. With the Eppendorf 96-Well Cell Culture Plate the edge effect can be reduced to a minimum. This application note compares the performance of Eppendorf 96-Well Cell Culture Plates with 96-well cell culture plates from different manufacturers concerning well-to-well variability in evaporation.* Introduction Cell-based assays are of increasing importance in almost all fields of biological science. The significance and value of the obtained data relies on the consistency of the performed assays. There are many sources of assay inconsistency in multi-well plates. Some of these aspects can be avoided by optimizing routine procedures in the laboratory. Possible measures include the implementation of adequate mixing and dissociation of the cells, the prevention of air bubble formation during cell seeding and the determination of optimal growth conditions concerning media formulation and seeding density. However, the edge effect can rarely be addressed by these precautions. As well-to-well variability mainly affects the peripheral wells of a plate this phenomenon is called the edge effect. The causes of the edge effect are complex. Temperature differences across the plate and evaporation effects in the edge wells during incubation have been described as two possible influencing factors. It is assumed that evaporation in the outer wells may lead to an accumulation of medium components (e.g. salts), thereby affecting cell metabolism. Varying cell responses may also be the result of temperature gradients. Both factors evaporation and temperature gradient may lead to higher variances between individual assays, thus resulting in unreliable data. There are different methods to prevent the edge effect. One approach to minimize the impact of a temperature gradient is the pre-incubation of the plate with freshly seeded cells at room temperature which results in a more even cell distribution and adhesion pattern. However, the edge effect does not only affect consistent cell growth. Even the most homogeneous monolayer may lead to unreliable results. Depending on the cell type used, pre-incubation at room temperature must be considered very carefully because some cell types are particularly sensitive to temperatures below 37 C. For applications requiring long-term incubation, instruments like humidity- and temperature-chambers are available. Their purpose is the creation of a constant micro-environment by reducing evaporation to a minimum and achieving almost consistent humidity and temperature levels. Probably the most common method to circumvent the edge effect is to avoid seeding cells in the peripheral wells. Not using the outer wells of a 96-well plate, however, reduces the number of wells for analysis to 60 and results in a 38 % lower throughput per plate. This method may avoid the edge effect but requires higher investments for consumables, incubator space, and time. Material and methods Preparation of plates For evaluating evaporation in 96-well plates the Eppendorf Cell Culture Plate as well as three plates from other manufacturers (A, B, and C) were tested (n=2). The Eppendorf Plate has a surrounding moat that can be filled with liquid (e.g. sterile water or PBS), thus insulating the edge wells. A B Fig. 1: The Eppendorf 96-Well Cell Culture Plate, filling options. A) Outer moat filled with liquid to insulate specifically the edge wells B) Insulation of all 96 wells by filling the complete inter-well space Your local distributor: Eppendorf AG Hamburg Germany eppendorf@eppendorf.com

12 PAGE 4 (BN 42) JANUARY 2015 Eppendorf 96-Well Cell Culture Plates: a Simple Method of Minimizing the Edge Effect in Cell-Based Assays Eppendorf Cell Culture Plate Manufacturer A Evaporation % evaporation [%] A B C D E F G H Evaporation % evaporation [%] A B C D E F G H Manufacturer B Manufacturer C Evaporation % evaporation [%] A B C D E F G H Evaporation % evaporation [%] A B C D E F G H Fig. 2: Comparison of evaporation in different 96-well plates. Liquid loss inside each well was determined after 5 days of incubation under standard cell culture conditions. Additionally it is possible to fill the complete inter-well space with liquid (Fig. 1). This ensures a higher temperature stability during incubation and slower cooling of the media in the wells when the plate is handled outside the incubator for a length of time, e.g. during microscopy. Prior to cell seeding, the plate with the filled moat and/or interwell space should be equilibrated in the incubator. Evaporation measurement The wells of the plates and where possible the space between the wells were filled with liquid, equilibrated and then incubated under standard cell culture conditions for 5 days. Liquid loss in each individual well was determined using crystal violet standard solution and absorbance measurement in the Eppendorf PlateReader AF Results and discussion Without insulation the edge wells of the Eppendorf 96-Well Cell Culture Plates show an average evaporation of only 1.8 % after 5 days of incubation under standard cell culture conditions. The low evaporation independent of the moat filling is probably a result of the optimized fit between lid and plate. Filling the surrounding moat of the Eppendorf plate with liquid further reduces evaporation to less than 1 %. The chimney-well design of the Eppendorf plate allows additional filling of the inter-well space minimizing evaporation in the edge wells to only 0.3 %. The design of the Eppendorf Cell Culture Plate makes the filling easy and convenient in just one pipetting step. A comparison of different 96-well cell culture plates shows that the Eppendorf Cell Culture Plate is the only plate that effectively minimizes evaporation (Fig. 2). Manufacturer A offers an option of partly insulating the peripheral wells but still shows an edge effect although more moderate compared to manufacturers B and C. Filling the complete inter-well space of the Eppendorf Cell Culture Plate with liquid contributes to a more homogeneous humidity and temperature stability within the plate. This leads to a decrease in evaporation and condensation and therefore reduces the edge effect. *The complete Application Note 326 is available for download at Tip! Watch the Video Minimizing the edge effect in cell-based assays Readers service Eppendorf Cell Culture Consumables Ref. no. 270 Your local distributor: Eppendorf AG Hamburg Germany eppendorf@eppendorf.com

13 (BN 42) JANUARY 2015 PAGE 5 Straightforward PCR Optimization and Highly Flexible Operation on the Dual Block Thermocycler Mastercycler nexus GX2 NILS GERKE AND ANDREA HELLBERG, EPPENDORF AG, HAMBURG, GERMANY Abstract The combination of a 64-well and a 32- well block in the Mastercycler nexus GX2 provides the user with the utmost flexibility. It offers optimum utilization of both thermoblocks, matching the needs of variable PCR sample throughput and additionally may be employed for routine applications while simultaneously performing PCR optimization using the temperature gradient function. The PCR applications presented here show that these flexible possibilities go hand in hand with consistent, high quality results. Introduction In many laboratories, especially those where multiple users rely on one instrument, flexible application options are the basic condition for optimized use of laboratory equipment and may increase the frequency of use. As PCR can take up a significant portion of time in the total workflow, efficiency in routine laboratories with multiple different applications can be similarly increased. This application note will present the extraordinarily flexible application possibilities of the dual block thermocycler Mastercycler nexus GX2: > > PCR optimization via temperature gradient function > > Reproducibility and comparability of PCR performance on the 64-well and 32-well block > > Thermal independence of both thermoblocks Materials and methods The 64-well block has a temperature gradient function across 8 positions in horizontal orientation. A GC-rich fragment (484 bp) of the human β-actin gene (ACTB) was chosen for amplification in this study. For detailed information on procedures, reagents, and instruments used, please see Application Note 289 which is available at applications. 1) PCR optimization via gradient function An initial temperature gradient run was performed to determine a suitable annealing temperature (range used: C), followed by a second gradient run to determine the optimum denaturation temperature (range used: C; all other conditions were identical). 2) Reproducibility and comparability Three PCRs were run in parallel on the 64-well and 32-well block using the conditions optimized under 1). The blocks were loaded with 12 samples each, distributed across the entire respective block (Fig. 1) Fig. 1: Positioning of the 12 PCR samples on the 64-well and 32-well block, respectively The PCR product concentration was determined on the Agilent 2100 Bioanalyzer using the Agilent DNA 1000 kit. 3) Thermic independence of both thermoblocks To test thermic independence, a PCR run with 12 samples was performed on the 64-well block using the conditions described above, with the 32-well block under a continuous holding temperature of 4 C. Subsequently, a second run with 12 samples was performed on the 32-well block while a temperature of 99 C was continuously held on the 64-well block. Results and discussion 1) PCR optimization via gradient function The gradient function of the Mastercycler nexus GX2 enables optimization of the annealing as well as the denaturation temperature. Analysis of PCR products from the annealing temperature gradient run clearly showed that low annealing temperatures led to formation of non-specific products above 500 bp and at ca. 350 bp (Fig. 2A). For this reason, an annealing temperature of 64 C was selected for the following runs. The analysis of the second gradient run revealed the formation of non-specific products at a lower denaturation temperature of ca. 93 C (Fig. 2B). A B M M Fig. 2: PCR products resulting from the gradient runs for determination of (A) annealing and (B) denaturation temperatures, respectively. The appropriate gradient temperatures [ C] of both runs are inserted at the bottom of the gel image (M: Length marker, GeneRuler 50 bp DNA Ladder, Thermo Fisher Scientific ) Your local distributor: Eppendorf AG Hamburg Germany eppendorf@eppendorf.com

14 PAGE 6 (BN 42) JANUARY 2015 Straightforward PCR Optimization and Highly Flexible Operation on the Dual Block Thermocycler Mastercycler nexus GX2 A 64-well block 32-well block C M M M M B D [ng/µl] M M well block 32-well block Fig. 3: (A C) Gel images of the PCR products resulting from the triplicate repeat PCR runs on the 64-well block and 32-well block; (D) Arithmetic means ± standard deviations of the PCR product concentration of the triplicate repeat PCR runs: light gray bar corresponds to gel A, medium gray bar to gel B, and dark gray bar to gel C on the 32-well and 64-well blocks. The blue bar displays the overall arithmetic mean ± standard deviation of the respective block. At higher temperatures, only the specific product associated with a higher yield was observed. Thus, 97 C was determined as the suitable denaturation temperature for subsequent work. 2) Reproducibility and comparability Triplicate PCR runs, each with 12 samples, served to verify the reproducibility of the PCR results obtained from each block, as well as the comparability between the PCR products resulting from the 64-well and 32-well blocks, respectively (Fig. 3A C). Analysis of the PCR product concentration from the 36 samples shows that product yields are reproducible on the 32-well block (overall average concentration = ng/µl; standard deviation = 2.5 ng/µl) as well as the 64-well block (overall average concentration = ng/µl; standard deviation = 2.95 ng/µl) across all three runs (Fig. 3D). These results further highlight not only consistent quality of results within one block, but also a high degree of consistency between the 32-well and the 64- well block. 3) Thermic independence of both thermoblocks The PCR results from both runs (Fig. 4) showed no evidence of external thermic influence across all positions in either of the Mastercycler nexus GX2 blocks, even when the neighboring block was programmed to a permanently extreme high or low holding temperature. Conclusion In addition to the independent use of two thermoblocks, the user may take maximum advantage of the new Mastercycler nexus X2 and its asymmetrical 64-well and 32-well block formats to accommodate different sample numbers. Therefore, the Mastercycler nexus X2 provides the user with the utmost flexibility while simultaneously delivering high quality results. This underscores the instrument s specific suitability for laboratories in which multiple users rely on one thermocycler, as well as for routine laboratories performing numerous different PCR reactions of low to medium sample number. This flexibility can be further expanded by using the Mastercycler nexus GX2 which offers a temperature gradient function for PCR optimization. A 1000 B Readers service Fig. 4: (A) 12 PCR products of the 64-well block during a permanent holding temperature of 4 C on the 32-well block; (B) 12 PCR products of the 32-well block during a permanent holding temperature of 99 C on the 64-well block Mastercycler nexus X2 Ref. no. 272 Your local distributor: Eppendorf AG Hamburg Germany eppendorf@eppendorf.com

15 (BN 42) JANUARY 2015 PAGE 7 Performance Comparison of Eppendorf Conical Tubes: Cap Tightness, Centrifugation Stability, and Leachables Levels RAFAL GRZESKOWIAK AND KATJA KAROW, EPPENDORF AG, HAMBURG, GERMANY Introduction Conical tubes with screw cap belong to most commonly used laboratory tube formats and are used in many laboratory procedures. Typically they have to withstand a great variety of conditions often regarded as extreme: temperatures between 86 C and 100 C, high centrifugal forces, aggressive chemicals or solvents, and many others. Under such broad range of conditions the tube has to remain stable and the screw cap tightly sealed ensuring protection of samples, user, and equipment. Furthermore, as recent scientific evidence suggests, plastic consumables can release various substances and hamper many experimental procedures [1]. Absence or very low levels of such substances in laboratory tubes may therefore be crucial for most applications and increase data quality and process safety in general. Here we investigate the performance of Eppendorf Conical Tubes 15 ml and 50 ml under application-relevant conditions and compare them to conical tubes of other manufacturers. We demonstrate that Eppendorf Conical Tubes as compared to other common brands provide excellent combined performance in these safety tests and show minimal levels of leachables. Materials and methods Centrifugation stability Two 15 ml and 50 ml conical tubes each of different manufacturers (Eppendorf, Fa, C, and G) were filled to 2/3 volume with water:phenol:chloroform (2:1:1) mixture. The tubes were centrifuged at 18,000 x g for 30 min at 4 C and 40 C (Eppendorf Centrifuge 5810 R, rotor FA ). After centrifugation the tubes were visually inspected. Screw cap tightness Conical tubes of different manufacturers (Eppendorf, Fa, C, and G) were filled with 96 % ethanol to their maximal volume and subsequently weighed. The tubes were then horizontally stored in a freezer at 86 C for 24 hours. After storage the tubes were equilibrated to room temperature and weighed using an ultra-precise scale. Sample loss was calculated as percentage of starting weight. Leachables spectrum Eight 15 ml and four 50 ml conical tubes each of different manufacturers (Eppendorf, C, T, Fi, S, and Fa) were filled to 90 % of nominal volume with pure water. The tubes were incubated in the Eppendorf ThermoMixer comfort at 90 C, 600 rpm for 30 min. Following incubation absorbance spectra were obtained (between 220 nm and 340 nm) using the Eppendorf BioSpectrometer and Eppendorf UVette cuvettes. The graphs depict average curves of three independent measurements. The dsdna concentration was calculated from the extinction at 260 nm using the factor 50 μg/ml per unit of extinction. Results and discussion Centrifugation stability Higher speed centrifugation of samples with organic solvents is challenging for bigger laboratory vessels. Particularly at lower temperatures conical tubes may show deformation/cracks of the tube wall or leakage of the screw cap, which results in sample loss and contamination. Manufacturer Eppendorf Manufacturer Fa Manufacturer C Manufacturer G Table 1 Conical Tubes 15 ml and 50 ml No damage Tube wall/lid damage Tube wall/lid damage No damage In our test a typical application of nucleic acid extraction was simulated by centrifuging the tubes at 18,000 x g with a water:phenol:chloroform (2:1:1) mixture. The results shown in Table 1 confirm high centrifugal stability of Eppendorf Conical Tubes as well as tubes from manufacturer G. All other vessels exhibited major wall/lid deformation and cracks, and all or at least the majority of the content was spilled in the rotor. Screw cap tightness Long term storage at very low temperatures and/or use of volatile samples with organic solvents may lead to sample loss. In our test ethanol samples were stored in horizontal position at 86 C to increase vapor pressure and lid strain under ultra-low temperature conditions. Fig. 1 shows sample loss after 24 h storage in different conical tubes. Both 15 ml and 50 ml Conical Tubes from Eppendorf showed lowest relative sample loss under the conditions tested. Leachables spectrum Recent scientific evidence indisputably shows that various additives (leachables) used in the production process of consumables may hamper various experimental systems and falsify the results [2]. To date many such substances have been identified and a routine method of absorption spectra measurement has been established as indication of the general amount of substances leached from the given consumable. Your local distributor: Eppendorf AG Hamburg Germany eppendorf@eppendorf.com

16 PAGE 8 (BN 42) JANUARY 2015 Performance Comparison of Eppendorf Conical Tubes: Cap Tightness, Centrifugation Stability, and Leachables Levels 12 Sample loss in conical tubes A 0.18 Absorbance scan of water following incubation in different 15 ml conical tubes Sample loss in % Eppendorf G Fa C 15 ml 50 ml Absorbance Eppendorf Manufacturer C Manufacturer T Manufacturer Fi Manufacturer S Manufacturer Fa Fig. 1: Sample loss after horizontal storage of ethanol in different conical tubes at 86 C for 24 h. Sample loss is expressed as percentage of initial weight before storage. n 15 ml: 50 tubes from Eppendorf, 38 tubes each from G, Fa, C n 50 ml: 50 tubes from Eppendorf, 22 tubes each from G, Fa, C Wavelength [nm] Theoretical dsdna concentrations of water samples incubated in different conical tubes B Absorbance scan of water following incubation in different 50 ml conical tubes 0.07 Manufacturer Fa 0.06 Eppendorf Manufacturer S Manufacturer Fi Manufacturer T Absorbance Manufacturer C Manufacturer T Manufacturer Fi Manufacturer S Manufacturer Fa Manufacturer C Eppendorf 50 ml 15 ml dsdna concentration [µg/ml] Wavelength [nm] Fig. 3: Theoretical dsdna concentration in water samples after incubation in different 15 ml and 50 ml conical tubes for 30 min at 90 C. The concentrations were calculated from the extinction at 260 nm using the factor 50 µg/ml per unit of extinction. Fig. 2: Absorbance spectra of pure water following incubation in different conical tubes at 90 C for 30 min. A) 15 ml conical tubes B) 50 ml conical tubes In Fig. 2 we show such absorption spectra of water samples incubated at 90 C for 30 min in various conical tubes. In summary the data show the lowest levels of UV-absorbing substances for conical tubes (both 15 ml and 50 ml) from Eppendorf and manufacturer S. Absorption spectra of tubes from manufacturers Fa, Fi, and C were the highest under the conditions used. The absorbance values obtained at 260 nm translate to dsdna concentrations and this may yield false elevated results during photometric analyses of molecules such as nucleic acids and proteins which are primarily conducted at nm. Respective values of theoretical dsdna concentrations derived from absorbance at 260 nm are shown in Fig. 3. The lowest values were observed for samples incubated in Eppendorf and manufacturer S conical tubes. Manufacturers Fa, Fi, and C showed the highest values. Noteworthy are large concentrations differences (up to 5 fold) between 15 ml and 50 ml tubes of manufacturers C and Fa. These may indicate higher material inhomogeneity and different rates of leaching in these vessels. Conclusion In this application note we investigated key parameters of Eppendorf Conical Tubes 15 ml and 50 ml under application-relevant conditions and compared to conical tubes of other manufacturers. The examinations focused primarily on parameters which have a high significance for many applications: centrifugation stability, screw cap tightness, and leachable levels. The comparative evaluation of the Eppendorf Conical Tubes 15 ml and 50 ml demonstrates excellent combined performance in all tests and shows minimal levels of leachables making them ideal vessels for a wide array of demanding applications. Literature [1] McDonald GR, Hudson AL, Dunn SM, You H, Baker GB, Whittal RM, Martin JW, Jha A, Edmondson DE, Holt A. Bioactive contaminants leach from disposable laboratory plasticware. Science 2008; 322:917. [2] McDonald GR, Kozuska JL, Holt A. Bioactive Leachates from Lab Plastics. G.I.T. Laboratory Journal Europe 2009; 13: Readers service Eppendorf Conical Tubes 15 ml and 50 ml Ref. no. 276 Your local distributor: Eppendorf AG Hamburg Germany eppendorf@eppendorf.com

17 NO COMPROMISES FROM 0.5 ML TO 50 ML! INNOVATION 9 NILS GERKE, EPPENDORF AG No Compromises from 0.5 ml to 50 ml! Tip And what do YOU think of us? Another step in the development of Eppendorf Tubes has been accomplished. With the new Eppendorf Conical Tubes 15 ml and 50 ml Eppendorf Tubes now cover the entire spectrum of volumes between 0.5 ml and 50 ml. The Conical Tubes meet the highest demands of diverse laboratory applications. Safe design and easy handling ensure excellent sample processing. BioNews has been published by Eppendorf since From a modest brochure in half-letter format and initially produced only irregularly, our popular customer magazine has emerged. It comes out twice per year and is distributed globally. Convincing high quality! Eppendorf Tubes 0.5 ml; 1.5 ml; 2.0 ml; 5.0 ml, and now 15 ml and 50 ml! Optimized handling Frequently it is the supposedly small details which will make a difference. The special attention of our developers was directed towards the design of the screw cap. The grooved sides with multiple flat sections not only minimize rolling off the workbench; more importantly, they enable a stable, slip-free grip, allowing secure opening and closing with only one hand. The new Eppendorf Conical Tubes 15 ml and 50 ml: optimally suited for centrifugation, mixing, and sample storage Expanded purity grade The new Conical Tubes feature an expanded purity grade: they are not only sterile and pyrogen-free; in addition they are free of DNases and RNases as well as human and bacterial DNA. This makes them ideal for cell biology applications in a sterile environment, as well as for laboratory protocols in both microbiology and molecular biology, where freedom from contamination is paramount. Versatile, e. g. in the rotor FA of the Centrifuges 5810 / R and 5804 / R Versatile The utmost level of manufacturing precision and robustness guarantee trouble-free use of the tubes in laboratory instruments such as centrifuges and thermomixers, thus contributing significantly to high quality, reproducible results sample by sample. Additional information is available at or in the latest brochure which you may order by reference number. Eppendorf Conical Tubes 15 ml and 50 ml Ref. no. 276 Naturally, BioNews was subject to one or the other facelift over the years, both optically and with respect to content. The steadily increasing number of subscribers confirms to us that we are still on the right track with our concept, even in our 22 nd year. Of course, we could now lean back in comfort, but we are curious! > > Curious about your opinion! > > Curious about your reading habits! > > Curious to learn how we can become even better in order to continue to support you with relevant information in your daily laboratory life. Participate in our online survey! In order to receive your feedback on the questions outlined above we are inviting you to participate in a short online survey starting February 1, With a little luck you could win one of ten Eppendorf pipettes which will be raffled off among all participants. The rules for the prize draw can be found at No recourse to legal action. We are looking forward to your participation!

18 10 STRAIGHT FROM THE LAB FINEST PRODUCTS FOR FOOD AND BEVERAGE LABS KAY KÖRNER, EPPENDORF AG Finest Products for Food and Beverage Labs Nourishing the world in the 21st century requires new ideas from drought-tolerant crops with high protein yields to streamlined processes in food production and quality control. Nurturing ideas to grown-up solutions requires determination, passion, and excellent tools. Eppendorf s tools for the laboratory have been among the finest and best for more than 60 years. Let us sort out the details of your daily lab challenges so you have the peace of mind to focus on the science of the food of tomorrow. Until the year 2100 there will be ten billion people living on this planet, according to UN estimates. One of the biggest challenges for the 21 st century is feeding this steadily increasing population. Crop yields for corn, rice, wheat, and soy would have to double during the coming 35 years. Due to the fact that even today arable land is scarce and getting scarcer, food research all over the world has to pave the way to nutrient-rich and droughttolerant crops. As a result of a steady increase in consumption of meat and dairy products, an even higher production of animal feed is required and crop demand is therefore rising dramatically. Food production Downstream processing of food has to be optimized to minimize nutrient loss. Nutrient levels have to be tested before and after new processing steps. Heating, cooling, packaging everything can have an influence on the quality of food and beverages. Food research The amount of arable land is not growing with the world population. Thus, applied research has to show ways to increase nutrient levels in crops, optimize crop rotation, or make crops more droughttolerant. The seasonal crop samples needed for this research are invaluable and must be processed fast and professionally.

19 FINEST PRODUCTS FOR FOOD AND BEVERAGE LABS STRAIGHT FROM THE LAB 11 Food analysis Food analysis and food quality control require reproducible workflows in every detail. Only the best lab products give you the confidence in the data you need. Your results can make a big difference. The development of modern food safety testing procedures is extremely important and ongoing. A big milestone was the implementation of the Food Safety Modernization Act in the US in 2011 as it will lead to state-of-the-art food testing that will result in a push for modernization in food testing laboratories all over the world. Eppendorf s user-friendly and reliable products are designated to help with these future challenges. Example: pipetting In food analysis, the primary material can come from a vast variety of sources and bring along a lot of unique challenges. Cooling, heating, mixing, centrifugation all or any of these steps can be necessary. The one thing that combines all these steps is the pipette. Eppendorf s electronic pipettes like the Eppendorf Xplorer are optimized for these processes. They allow fatigue-free pipetting and minimize pipetting errors. Where high sensitivity and reproducibility are essential, ep Dualfilter T.I.P.S. build the perfect system with Eppendorf pipettes for contamination-free pipetting by practically 100 % retention of aerosols and biomolecules. Strain selection and media optimization Whether searching for advanced yeasts in brewery, proper bacteria in dairy industry, or a powerful production strain for nutrition supplements the DASbox is the superior tool for screening. Small working volumes, parallel operation of twelve and more bioreactors, and applying Design of Experiments (DOE) allow for fast, reliable, and cost-effective processing. Pilot and production Production processes in the food and beverage industries are multifaceted and constantly evolving. The modular design of the Eppendorf stainless steel fermentors facilitates these process requirements by allowing the addition or removal of options at any time at an impressive volume range up to 2,400 L. Close-up Eppendorf μcuvette G1.0 The Eppendorf μcuvette G1.0 is a microliter measuring cell specially developed for measuring high concentrations in the smallest volumes. It has an optical path length of only 1 mm which is ten times shorter than the light path length of standard cuvettes. Benefit: nucleic acid concentrations of volumes down to 1.5 µl can be measured with high reproducibility in a much broader concentration range without prior dilution. The μcuvette G1.0 has been exclusively developed for use in the Eppendorf BioPhotometer (all models since 1998) and the Eppendorf BioSpectrometer (all models). Eppendorf Xplorer 8-channel pipette with ep Dualfilter T.I.P.S. filtertips Stainless steel fermentor BioFlo Pro: 32 2,400 L working volume Additionally, Eppendorf offers a wide range of centrifuges, mixers, ULT freezers, CO 2 incubators, reaction vessels, and laboratory automation systems. You will find more details at or in the new brochure Delicious Details. Close-up: The hydrophobic surface coating on quartz glass is a particularly innovative feature. It guarantees the precise formation and positioning of the sample volume and simple cleaning. Due to the spherical drop shape the Eppendorf µcuvette G1.0 requires a smaller volume than comparable products. Besides the reddot Design Award 2013 the Eppendorf µcuvette also received the Forum LABO & BIOTECH Innovation Prize 2013 in the category Miniaturization. Besides pipettes and matching pipette tips Eppendorf offers intelligent solutions for many other applications in food industry laboratories: Brochure Delicious Details Ref. no. 278 Eppendorf μcuvette G1.0 Ref. no. 251

20 12 NEWS TRADITION CONTINUED: NEW THERMOMIXERS SIRKKA SCHEEFF, EPPENDORF AG Tradition Continued: New Thermomixers Tip User Test of TransferMan 4r For the past 50 years Eppendorf has set the standard in the fields of mixing and temperature control of small volumes. We continue this tradition by introducing two new thermomixers. The Eppendorf ThermoMixers F0.5 and F2.0 with their fixed blocks for 0.5 ml tubes, and 2.0 ml tubes, respectively, are the perfect choice for common routine applications. Established quality since 1964 With the introduction of the first mixer and thermostat models in 1964 Eppendorf set the standard in the areas of mixing and temperature control technology for small volumes. Since that time, this range of products has seen continuous development, allowing it to adapt to the increasing demands of our users. 2-in-1 instruments for mixing and temperature control steps The ThermoMixers F0.5 and F2.0 were developed specifically for the tube formats 0.5 ml and 2.0 ml. Their superior mixing results are based on the unique 2D Mix-Control-Technology, which has already proven its worth in the popular Eppendorf ThermoMixer C as well as in the MixMate. It guarantees efficient and reliable mixing of even the smallest volumes in seconds. Defined temperature keys enable quick selection of common temperature parameters and ensure time-saving and stress-free operation during routine applications. Want to avoid condensation? An ideal tool to achieve outstanding temperature control is the new optional Eppendorf ThermoTop. The heated lid with condens.protect technology protects reliably against the formation of condensation on the tube lid and walls. Further information For details on the new mixing professionals please see the current brochure or visit Tip: You work with different tube formats? In this case, the Eppendorf ThermoMixer C and the Eppendorf ThermoStat C are the instruments of choice, for which a broad palette of interchangeable Smart- Blocks is available. Watch the ThermoTop video! The study of genetically modified animals provides important insights into the gene functions or, by modeling human diseases, to either understand disease mechanisms or aid drug development. The main techniques to generate those animal models require micromanipulation devices, but usually in slightly varying set ups and with different settings for the respective techniques. Therefore many laboratories generating mouse or rat models prefer to have separate workstations for the two main techniques, nucleic acid injection into fertilized oocytes and embryonic stem (ES) cell transfer into early embryos. With the multifunctional manipulator TransferMan 4r, one workstation is sufficient for all applications because of its easy angle adjustment, vibration-free movements, and the new Eppendorf DualSpeed joystick. Ronald Naumann* and his team tested the TransferMan 4r performance for manipulation techniques routinely performed in his lab over an extended period of time. Ronald Naumann: With the new motor and joystick concept, the TransferMan 4r is a fantastic option for an all-in-one solution especially for those laboratories with only one injection stage. For full details see the Application Note No. 285 The Eppendorf TransferMan 4r, one manipulator for all genetic engineering techniques. Available for download at New: Eppendorf ThermoMixer F0.5 / F2.0 The ThermoTop with condens.protect technology ensures optimal reaction conditions. Eppendorf ThermoMixer F0.5 / F2.0 Ref. no. 277 *Transgenic Core Facility, Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany