Eppendorf Tubes 5.0 ml: The»Missing Link« No

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1 No Eppendorf Tubes 5.0 ml: The»Missing Link«(BN 40) JaNuarY 2014 PaGe 3 Quantification of Nucleic Acids Using a Factor-Based Method in the Eppendorf PlateReader AF2200 > Eppendorf Reference 2: The New Legend > New BioBLU 1-Liter Single-Use Bioreactors > Ergonomics in Today's Laboratory Routine TaNJa Musiol1, BeTTiNa scheumer 2, Markus lapczyna Introduction determination of concentration via a sample-specific extinction factor is a common approach for the cuvette format, but not for the plate format. The underlying calculation is lambertbeer's law: A = ε c d a = absorbance, ε = extinction coefficient, c = concentration, d = optical path length for this calculation, knowledge of the sample specific factor, as well as the exact path length are required. in the case of cuvettes, the path length is determined by the shape of the cuvette, as the light beam traverses the cuvette horizontally (fig. 1). Cuvette in this article it will be shown how, using the eppendorf Microplate uv-vis with a defined volume, the concentration of a dsdna solution is determined in the eppendorf Platereader af2200 by means of a factor-based method. since the exact dimensions of the plate and its wells are known, the software already contains the respective path lengths for pre-defined filling volumes. calculation of sample concentration is performed analogous to the familiar process employed for instruments using cuvettes. exact pipetting is of course a prerequisite to ensure uniform filling heights (fig. 2B). The results from the factor-based method are compared with results evaluated via standard curve and with cuvette measurements carried out in a reference spectrophotometer. Material > ultrapure Herring sperm dna solution (life Technologies ; cat. no ) d > Quartz glass cuvette with a 10 mm path length (Hellma analytics) Light path > reference spectrophotometer Fig. 1: light beam through the cuvette in the case of absorbance measurements in a plate, the light beam traverses the sample vertically (fig. 2a). The path length is in this case determined by the filling height. since the filling height is not only dependent on the volume but also on the geometry of the well, exact determination of the filling height is critical. A > eppendorf Microplate uv-vis with uv transparent foil bottom (eppendorf) > eppendorf Platereader af2200 (eppendorf) > Tris buffer: 0.1 M, ph 8.0 Method The following dna concentrations are prepared : 5, 10, 25, 50, 60, 80 and B Microplate during the course of factor-based measurements, absorbance values at the following wavelengths are determined: 260 nm, 280 nm and 340 nm. The value generated at 340 nm is taken into consideration as a reference value in order to minimize the influence of contamination. The value obtained at 280 nm is used to calculate the a260/ a280 ratio and to determine the purity of the dna. Pure dna shows a ratio between 1.8 and 2.0. lower ratios may indicate protein contamination. The absorbance measurement at 260 nm is used to calculate the dna concentration following lambert-beer's law. calculation of concentration is performed by the instrument's software by using the sample specific extinction factor of 50 for double-stranded dna (dsdna). Microplate d Light path d Volume 100 ng/µl. every dilution step is checked in a quartz glass cuvette in a spectrophotometer at a 260 nm wavelength. The dilution steps 5, 25, 50 und 100 ng/µl are used for preparing the standard curve (Platereader method»uv 260 nm with standards«). The concentrations 80 ng/μl, 60 ng/μl and 10 ng/μl are then determined via the standard curve. subsequently, all concentrations are again determined using the factorbased method (Platereader method»uv 260 nm with factor«) and compared to the results obtained with the standard curve, as well as with those obtained from the cuvette measurements on the reference spectrophotometer. all measurements (factor and standard) are performed with 100, 200, 300 µl (one plate for each volume). Light path Application Notes Comparative Run Time Evaluations of PCR Thermal Cyclers Quantification of Nucleic Acids Using a Factor-Based Method in the Eppendorf PlateReader AF2200 etc. eppendorf ag, HaMBurG, GerMaNY eppendorf instrumente GMBH, HaMBurG, GerMaNY Volume Fig. 2: Photometric determination in a microplate. The path length is determined by the filling height. A) different liquid levels due to unreproducible pipetting or high variances in the well geometry B) Photometric determination in plate readers requires identical liquid levels. Your local distributor: eppendorf ag Hamburg Germany eppendorf@eppendorf.com

2 2 Editorial Dear Readers Imprint Editorial team Berrit Hoff (Editor-in-Chief), Axel Jahns, Jochen Müller-Ibeler, Natascha Weiß Dear Readers, 50 years ago Eppendorf created the Eppendorf Tube and triggered a microtube evolution. Eppendorf Tubes are now used every day throughout the world and are a recognized standard in sample processing from 0.2 ml to 2.0 ml. While conical screw cap tubes (e. g. 15 ml tubes) are available for large volumes, up until now there has been no convincing solution for medium sample volumes. Here's the good news: The new Eppendorf Tube 5.0 ml now fills the gap between existing tube versions and enables the simple and safe processing of sample volumes up to 5.0 ml. As you may expect from Eppendorf, we also offer a comprehensive range of matching accessories for sample preparation and sample storage with numerous applicational benefits. Read more on pages 4 5! Publisher Eppendorf AG, Barkhausenweg 1, Hamburg, Germany Telephone: (+49) Fax: (+49) bionews@eppendorf.de Internet: 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. Do you already know about the new Multipette M4*? Its light weight and ergonomic design, which was optimized according to the most recent findings, as well as its intuitive operation enable simple, stress-free and ergonomic dispensing (page 6). Do you often have to pipette precious liquids? Are extraordinary precision and accuracy, a long service life, and an ergonomic design essential for your daily work? Then take a closer look at the new Eppendorf Reference 2, our premium pipette for your demanding work. This classic Eppendorf pipette with single-button operation is now also available as a multi-channel version (page 7). In addition to further articles on new products, this BioNews edition also includes detailed Application Notes and a new competition with great prizes. Enjoy reading! Your Eppendorf BioNews Editorial Team *US/CAN: Repeater M4

3 Contents 4 In the Spotlight Innovation Straight from the lab 7 11 Eppendorf Tubes 5.0 ml: The»Missing Link«4 5 Your Sample is Safe with ep Dualfilter T.I.P.S. SealMax 8»Smooth Operators«: New Micromanipulators 11 Ergonomics in Today's Laboratory Routine 10 A Really Cool Product Family: New Brunswick ULT Freezers 13 News / Tips Dispensing Newly Defined: Simple, Stress-Free, Ergonomic 6 Eppendorf Reference 2: The New Legend 7 Best Conditions for Your PCR 9 Correct Pipetting 10 New 1 Liter Vessels Expand the BioBLU Family 12 Prize Competition 14 Reply Fax / Readers' Service 15 Service (BN 40) JaNuarY 2014 PaGe 3 Quantification of Nucleic Acids Using a Factor-Based Method in the Eppendorf PlateReader AF2200 TaNJa Musiol1, BeTTiNa scheumer 2, Markus lapczyna eppendorf ag, HaMBurG, GerMaNY eppendorf instrumente GMBH, HaMBurG, GerMaNY Introduction determination of concentration via a sample-specific extinction factor is a common approach for the cuvette format, but not for the plate format. The underlying calculation is lambertbeer's law: A = ε c d a = absorbance, ε = extinction coefficient, c = concentration, d = optical path length for this calculation, knowledge of the sample specific factor, as well as the exact path length are required. in the case of cuvettes, the path length is determined by the shape of the cuvette, as the light beam traverses the cuvette horizontally (fig. 1). Cuvette in this article it will be shown how, using the eppendorf Microplate uv-vis with a defined volume, the concentration of a dsdna solution is determined in the eppendorf Platereader af2200 by means of a factor-based method. since the exact dimensions of the plate and its wells are known, the software already contains the respective path lengths for pre-defined filling volumes. calculation of sample concentration is performed analogous to the familiar process employed for instruments using cuvettes. exact pipetting is of course a prerequisite to ensure uniform filling heights (fig. 2B). The results from the factor-based method are compared with results evaluated via standard curve and with cuvette measurements carried out in a reference spectrophotometer. Material > ultrapure Herring sperm dna solution (life Technologies ; cat. no ) d > Quartz glass cuvette with a 10 mm path length (Hellma analytics) Light path > reference spectrophotometer Fig. 1: light beam through the cuvette in the case of absorbance measurements in a plate, the light beam traverses the sample vertically (fig. 2a). The path length is in this case determined by the filling height. since the filling height is not only dependent on the volume but also on the geometry of the well, exact determination of the filling height is critical. A > eppendorf Microplate uv-vis with uv transparent foil bottom (eppendorf) > eppendorf Platereader af2200 (eppendorf) > Tris buffer: 0.1 M, ph 8.0 Method The following dna concentrations are prepared : 5, 10, 25, 50, 60, 80 and B Microplate during the course of factor-based measurements, absorbance values at the following wavelengths are determined: 260 nm, 280 nm and 340 nm. The value generated at 340 nm is taken into consideration as a reference value in order to minimize the influence of contamination. The value obtained at 280 nm is used to calculate the a260/ a280 ratio and to determine the purity of the dna. Pure dna shows a ratio between 1.8 and 2.0. lower ratios may indicate protein contamination. The absorbance measurement at 260 nm is used to calculate the dna concentration following lambert-beer's law. calculation of concentration is performed by the instrument's software by using the sample specific extinction factor of 50 for double-stranded dna (dsdna). Microplate d Light path Light path d Volume 100 ng/µl. every dilution step is checked in a quartz glass cuvette in a spectrophotometer at a 260 nm wavelength. The dilution steps 5, 25, 50 und 100 ng/µl are used for preparing the standard curve (Platereader method»uv 260 nm with standards«). The concentrations 80 ng/μl, 60 ng/μl and 10 ng/μl are then determined via the standard curve. subsequently, all concentrations are again determined using the factorbased method (Platereader method»uv 260 nm with factor«) and compared to the results obtained with the standard curve, as well as with those obtained from the cuvette measurements on the reference spectrophotometer. all measurements (factor and standard) are performed with 100, 200, 300 µl (one plate for each volume). Volume Fig. 2: Photometric determination in a microplate. The path length is determined by the filling height. A) different liquid levels due to unreproducible pipetting or high variances in the well geometry B) Photometric determination in plate readers requires identical liquid levels. Your local distributor: eppendorf ag Hamburg Germany eppendorf@eppendorf.com Khandaker Siddiquee, Ma Sha A Novel Method for the Expansion of Mesenchymal Stem Cells Using the Eppendorf New Brunswick S41i CO2 Incubator Shaker 1 2 Tanja Musiol, Bettina Scheumer, Markus Lapczyna Quantification of Nucleic Acids Using a Factor-Based Method in the Eppendorf PlateReader AF Natascha WeiSS, Frauke Gotzhein Comparison of Eppendorf Tubes 5.0 ml to Conical 15 ml Tubes in Regard to Releasable UV-Absorbing Substances (Leachables) 5 6 Nils Gerke Comparative Run Time Evaluations of PCR Thermal Cyclers 7 8 3

4 4 IN THE SPOTLIGHT Eppendorf Tubes 5.0 ml: The»Missing Link«Berrit Hoff, EPPENDORF AG Eppendorf Tubes 5.0 ml: The»Missing Link«Many laboratory applications call for volumes which exceed the capacity of the standard 1.5 ml or 2.0 ml reaction tubes. For medium sample volumes between 2.0 ml and 5.0 ml users have traditionally resorted to working with vessels designed for much larger volumes, typically 15 ml conical screw cap tubes. With the new Eppendorf Tube 5.0 ml, Eppendorf now offers the»missing link«. pipettes, are used, which is less ergonomic, especially when working in a sterile hood. Last, but not least, our extensive selection of accessories, which allow the use of the tube in almost any context. Which material is used for the production of the 5.0 ml Tube? The Eppendorf Tube 5.0 ml closes the gap between microcentrifuge tube and conical screw cap vessel. An especially clear and high quality polypropylene. It offers high transparency for good sample visibility while simultaneously minimizing leaching. Therefore, the user does not have to worry about substances leaching from the plastic which may influence the experiment. In comparison with 15 ml conical tubes, the Eppendorf Tubes 5.0 ml do very well in this category (editor's note: please also refer to the Application Note on pages 5 6). In concert with a unique system of matching accessories for sample preparation and storage, a comprehensive and thought-out solution is now available. More details directly from Dr. Daniel Wehrhahn (Global Product Manager Consumables). You like to describe the Eppendorf Tube 5.0 ml as the»missing link«, even though tubes in this volume range already exist. DW: This is correct; however, these vessels often lack important features such as high centrifugation stability or a safe seal. This is why users mainly resorted to the tried and tested 15 ml conical tubes. What sets the new tube apart? For one, comfortable handling: thanks to its snap cap lid, our tube can be effortlessly opened and closed with one hand. The handling of screw cap tubes is less comfortable. Second, better protection against cross-contamination. Especially during sample processing of small volumes in large, conical tubes, the pipette cone is often inserted deeply into the tube in order to remove the entire sample, or supernatant above a pellet. In cell culture, where 15 ml tubes are often employed for passaging cells, longer serological pipettes, or Pasteur Furthermore, the tubes feature high chemical resistance as well as temperature stability and mechanical stability. How stable are the tubes? Eppendorf Tubes 5.0 ml were designed for safe and stable centrifugation up to 25,000 x g, thus preventing sample loss when performing quick protocols. This centrifugation stability also exceeds that of most 15 ml conical tubes! How about thermic stability? The temperature range of the Eppendorf Tubes 5.0 ml encompasses -86 C to 80 C. During incubation up to 100 C the»tube

5 Eppendorf Tubes 5.0 ml: The»Missing Link«IN THE SPOTLIGHT 5 Tip Eppendorf Tubes 5.0 ml systematically simple and safe Perfect mixing, heating and cooling with the Thermomixer C Optimized adapters for existing rotors Precise one-step pipetting with Eppendorf pipettes and ept.i.p.s. 5.0 ml Clip 5.0 ml«ensures that the tubes remain securely sealed. Which purity grades are available for the Eppendorf Tube 5.0 ml? In addition to the established purities Eppendorf Quality, PCR clean and Eppendorf Biopur we also offer the purity grade»sterile«. These products are additionally tested for the absence of pyrogens. Owing to the variety of certified purity grades, the 5.0 ml tubes are suited for all types of different methods within cell biology and molecular biology laboratories. Furthermore, the special Eppendorf LoBind material, offered in this format for the first time, makes them ideal for work with sensitive nucleic acid or protein samples. It is noticeable that the 5.0 ml Tube also features a conical shape. Integrating the tube seamlessly into the existing workflow, and ensuring its compatibility with instruments and accessories designed for 15 ml conical tubes which are already present in the laboratory was important to us. This way, the user can continue to use many adapters and racks in a cost-saving fashion. At the same time, we have optimized our own selection of instruments and acces- sories: we offer matching high-speed rotors for our Centrifuges 5430/R, 5427 R, 5804/R and 5810/R. In addition, adapters and inserts for all existing Eppendorf rotors with bores for 15 ml or 50 ml vessels are available. For temperature control and mixing, 5.0 ml interchangeable blocks for the Eppendorf ThermoMixer C and Thermomixer comfort, the ThermoStat C and the ThermoStat plus, are available. Even a 5.0 ml solution for the automatic pipetting system epmotion was included. For which methods are the Eppendorf Tubes 5.0 ml especially recommended? Larger volumes of culture media are advantageous for a multitude of applications. For example, when isolating certain types of plasmid DNA considerably higher yields may be achieved in fewer steps when the volume of the starter culture is increased to 5.0 ml in a larger tube [1]. You have been in charge of this project from the beginning. Do you see a parallel to the Eppendorf microliter system, which was launched in the early sixties? Well, even though we are only in the beginning stages of marketing, we notice very high demand by our users. Our»missing link«, and the holistic approach of our system, has obviously struck bull's eye. Considering all this, both projects are comparable. This validates and also pleases us! [1] APPLICATION NOTE No. 262/ June 2013 ( Eppendorf Tubes 5.0 ml Ref. no. 264 > > Simple, practical and ergonomic singlehand operation > > Hinged lid for minimized sample evaporation during storage and incubation in a wide range of temperature from 86 C to 80 C > > Exceptionally high-quality, transparent polypropylene, free of plasticizers, biocides or mold release agents, for reliable test results > > Large labeling area > > g-safe : maximum safety and stability for centrifugation up to 25,000 x g; fast and efficient protocols > > Comprehensive system accessories available: adapters, rotors, thermoblocks and storage boxes > > For guaranteed purity: batch certified in the qualities PCR clean, Sterile and Eppendorf Biopur > > High resistance to chemicals and mechanical strain > > Available in Eppendorf material for maximum recovery of valuable samples Eppendorf offers a complete solution for centrifugation, heating and mixing, as well as liquid handling and storage in Eppendorf Tubes 5.0 ml. More information at

6 6 News Dispensing Newly Defined: Simple, Stress-Free, Ergonomic Sabine Kühn, Eppendorf AG Dispensing Newly Defined: Simple, Stress-Free, Ergonomic The new Multipette M4 (US/CAN: Repeater M4), the successor model to the popular Multipette plus (US/CAN: Repeater plus), is the ideal precision instrument for conducting long pipetting or dispensing series. Thanks to the principle of direct displacement, and a broad selection of Combitips advanced dispensing tips, the Multipette M4 masters even the most challenging dispensing problems with style. Its low weight, the design optimized according to the latest state of knowledge and its intuitive handling ensure simple, stress-free and ergonomic dispensing. Swift and stress-free work The new Multipette M4 facilitates and accelerates the performance of long pipetting series. Reliable system for sophisticated challenges In combination with the dispensing tips Combitips advanced, the Multipette M4 functions according to the direct displacement principle. The liquid is dispensed without an air cushion, in direct contact with the Combitip's piston. Whether you work with viscous solutions (e.g. glycerol), liquids with high vapor pressure (e.g. ethanol) or other problematic substances: independent of density, viscosity or volatility of the liquid, the correct volume is dispensed every time. Accurately, swiftly and free from contamination. The Multipette M4 makes a considerable contribution to the simplification as well as more rapid completion of long pipetting series, be it for aliquoting, when using kits, or for filling reaction tubes and plates. This way, one filling of the Combitip may be dispensed in up to 100 individual steps a comfortable solution for filling 96-well plates, to name just one application. The new integrated step counter displays the dispensing steps completed. This feature ensures error-free dispensing in the face of work interruptions or distractions. Speaking of work interruption: The new sleep function turns the Multipette M4 off when not in use, thus ensuring energy consumption that is easy on the battery! Combitips advanced: the perfect system component The Multipette M4 offers a broad volume spectrum, dispensing in steps from 1 μl up to 10 ml. Everything under control: dispensing volume and number of steps The right tip for each application: Combitips advanced are available in 9 volume sizes and 3 purity grades. To this end, Combitips advanced are available in 9 sizes from 0.1 ml to 50 ml. Even the variety of volumes leaves no wish unfulfilled: each Combitip allows 20 different dispensing volumes. Following insertion of the Combitip, the Combitipsize is recognized by a sensor. The dispensing volume selected via the volume selection wheel appears automatically on the display. After use, the Combitip advanced is simply ejected and disposed of by onehanded button operation, without any contact or danger of contamination. Learn more All details are available at or in the current brochure (simply order by reference number). Multipette M4 Ref. no. 268

7 Eppendorf Reference 2: The New Legend News 7 Christiane Markau, Eppendorf AG Eppendorf Reference 2: The New Legend Reliability, longevity, extraordinary precision and accuracy, paired with the special single-button handling concept. These features have led the Eppendorf Reference pipette to success and turned it into a legend. Now the bar has been raised even higher for the development of our new premium pipette Reference 2. The result: modern design, reduced weight and operating forces, and with multi-channel variants the largest selection of varieties for an Eppendorf pipette. Eppendorf Reference 2: the new reference class for modern liquid handling tools Single-button operation is the hallmark of the Reference 2. Volume selection, liquid aspiration and dispensing, as well as tip disposal, occur with just one button fast and ergonomic. The pipette achieves quick tip ejection with active aerosol reduction. No lateral movement of the thumb is required for tip ejection, thus reducing the risk of repetitive strain injuries (RSIs). NEW! Improved traceability Security is provided by the integrated RFID chip, on which important pipette data such as serial number and factory calibration dates are irrevocably saved. Using the Eppendorf TrackIT RFID reader, these data can be read and also supplemented with additional information such as inventory number, calibration due date and location. The Eppendorf TrackIT software supplies an overview of all data entered and optionally saves each change in a defined location where it may be read into a LIMS system. In addition, the serial number of the Reference 2 is printed on both the upper and the lower parts of the pipette. This way, replacement or exchange of the lower part becomes immediately obvious. Reproducibly high quality results The spring-loaded tip cone ensures low operating and ejection forces, as well as reproducible, user-independent tip fit. For the new multi-channel variants, the new channel indicator guarantees a continuously identical pipette alignment throughout the work process. A premium pipette for highest expectations Only state of the art technologies and select materials have been introduced into the Reference 2. It is completely autoclavable, it offers high reproducibility and reliability. It is therefore the ideal instrument for working with valuable liquids, as well as for all applications requiring maximum accuracy. Endurance test for the new legend The new Reference 2 is as long lived and robust as its predecessor. The proof is delivered by our short film»the New Legend: The outstanding robustness of the Eppendorf Reference 2 pipette«. It shows the Reference 2 pass a very special endurance test. The film and further information are available at We would be pleased to send you the detailed brochure; just order by reference number. The name»reference«stands for extraordinary precision and accuracy, a long service life, and an ergonomic design. Eppendorf Reference 2 Ref. no. 269

8 8 Innovation YOUR SAMPLE IS SAFE WITH EP DUALFILTER T.I.P.S. SEALMAX BRIGITTE KLOSE, EPPENDORF AG Your Sample is Safe with ep Dualfilter T.I.P.S. SealMax The prevention of contamination is essential in life-science research. For this reason, to protect pipettes and samples against foreign DNA or other substances, filter tips are mainly used. Filters with specific pore configurations are designed to protect the tip cone against aerosol contact and prevent cross contamination with the sample. But, in the case of accidental over-pipetting, liquid can penetrate through the filter and thus contaminate the tip cone. Therefore, some filter tips also provide protection against liquids. The filters for these tips contain sealing or barrierforming additives. As soon as they come into contact with the sample liquid, the filters swell and prevent the liquid from passing through the filter. The ability to recover as much sample as possible thereafter is questionable and often it cannot be excluded that the filter material, after having been in contact with the sample, may negatively affect the efficiency of classic PCR and quantitative real-time PCR.»SealMax«stands for maximum protection against contamination and sample loss The new violet/white filter of the ep Dualfilter T.I.P.S. SealMax filter tips combines reliable pipette protection against contaminating contact with the sample due to accidental over-pipetting with virtually 100 % protection against aerosols and biomolecules. This innovative filter is the first Dualfilter which immediately closes when it comes into contact with the sample, without inhibiting it. Sample loss is thus avoided when working with aqueous solutions having physical properties like water. In these cases the pipetting procedure can continue unimpeded without wasting time. Virtually all of the aspirated aqueous sample material can be recovered. Two layers for effective measurement The violet/white filter which is made of flexible material also has different pore sizes in its two layers for retaining aerosols and biomolecules. Like the established ep Dualfilter T.I.P.S. with a blue/white filter, the air flow rate is not affected. The filter penetration rate was measured according to EN 1822 with 99.5 % retention of aerosols, which Sample recovery 100 % 90 % 80 % 70 % 60 % 50 % 40% 30 % 20 % 10 % 0 % 10 µl Filter tip 200 µl Filter tip ep Dualfilter T.I.P.S. SealMax ep Dualfilter T.I.P.S. SealMax Ref. no. 262 Competitor G complies with HEPA (High Efficiency Particulate Airfilter) class E12. A study carried out by Eppendorf AG determined the sample recovery rate of ep Dualfilter T.I.P.S. SealMax and other self-sealing filter tips following over- pipetting of water. The recovery rate was significantly higher than those of the competitor products tested (Fig. 1). More information ep Dualfilter T.I.P.S. SealMax are available in the tested purity grade PCR clean/sterile (sterile and pyrogen-free) in volume ranges from 10 µl to 1,000 µl. Batch-specific certification is conducted by an independent laboratory. You can request the new brochure quoting the reference number or you can inform yourself online at Competitor A Fig. 1: A comparison of the sample water recovery rate of ep Dualfilter T.I.P.S. SealMax with the self-sealing filter tips of two competitors. The figure shows the average sample recovery rates in % collected from 10 individual measurements. Error bar = standard deviation. Source: Application Note 273.* Determination of the sample recovery rate of self-sealing filter tips in case of over-pipetting of water. *Available for download as a PDF at

9 (BN 40) JanuarY 2014 PAGe 1 A Novel Method for the Expansion of Mesenchymal Stem Cells Using the Eppendorf New Brunswick S41i CO 2 Incubator Shaker Khandaker Siddiquee & Ma Sha, Eppendorf, Inc., Enfield, CT, USA Introduction The expansion of stem cells, including mesenchymal stem cells (MSCs), has been successfully demonstrated using microcarrier-based small bioreactors such as spinner flasks. In this study, we explored a simple alternative to the MSC expansion in spinner flasks using conventional shake flasks in a CO 2 incubator with built-in shaking capability: the Eppendorf New Brunswick S41i. Here we compared microcarrier-based 12 days expansion of adipose-derived mesenchymal stem cells (AdMSCs) in spinner flasks to shake flasks analyzing parameters like cell growth, metabolism as well as quality and differentiation abilities of the cells. The New Brunswick S41i CO 2 Incubator Shaker, designed for both non-adherent and adherent cell culture applications, combines the precise temperature and CO 2 control of an incubator with the reliable New Brunswick laboratory shaker drive mechanism. Key features include sealed inner/outer doors, high-temperature disinfection, and reduced CO 2 consumption compared to competitor models [1]. Materials and methods AdMSCs were initially seeded in both systems at a density of 3 x 10 3 cells/cm 2 in appropriate medium. For the initial attachment of cells on the microcarriers (0.5 g of micron) in both systems, the agitation speed of the New Brunswick S41i CO 2 incubator shaker and rotation speed of the spinner were both kept at 50 rpm and incubated for 2 h at 37 C with 5 % CO 2. After that the volume was adjusted to 50 ml total volume and the speed in both systems was raised to 70 rpm. Cell culture studies were conducted for 12 days and samples were collected for cell growth, biochemistry and metabolite analysis daily. Cells on microcarriers were counted by a hemocytometer. The supernatants collected during cell counting were used for further biochemistry and metabolite measurements by an automated biochemistry analyzer. To assess the quality of AdMSCs after expansion and to confirm that the stem cell markers were retained during the microcarrier-based culture, CD44 and CD90-specific fluorescent immunoassays were performed. To prove their ability to differentiate, adipocyte and osteocyte differentiation assays were performed. Adipocyte and osteocyte differentiated cells were identified by cell-type specific staining with either Oil red O or Alizarin red S. For more detailed information on procedure and instruments used please see Application Note 259 (document available online at applications.) Results and discussion Cell growth studies revealed that AdMSCs cultured under shake flask conditions achieved excellent growth during the 12 day batch culture (Fig. 1A). Biochemistry and metabolite analysis revealed that glucose concentrations A Cell number (10 4 )/ml C Lactate concentration (g/l) Growth curve of AdMSCs Day Concentration of lactate in AdMSC cultures Day decreased from 1.09 g/l to g/l (for shake flask culture) and g/l (for spinner culture), whereas lactate concentrations increased from g/l to g/l (for shake flask culture) and g/l (for spinner culture) after 12 days of culture (Fig. 1B &C). The higher glucose consumption and lactate production rate seen in the shake flask culture supports the finding that the stem cells grew at a faster rate under the shake flask conditions. Furthermore, during early growth phase (day 4); the amount of ammonium accumulated in spinner flask culture (2.4 mm) was 1.8-fold higher than in shake flask culture (1.3 mm) (Fig. 1D). It has been shown that even low levels of ammonium (1.9 mm) inhibit MSC growth. The spinner culture has shown ammonium levels exceeding 2 mm early and throughout the culture process, which indicates the slower growth by the spinner method could be a result of ammonium toxicity-induced growth inhibition. The fact that the spinner culture had elevated ammonium levels early in the culture (not seen in the shake flask) also indicates possible stem cell damage B Glucose concentration (g/l) D NH 3 Concentration (mm) Concentration of glucose in AdMSC cultures Day Concentration of NH3 in AdMSC cultures Day Fig. 1: Analysis of AdMSCs growth and metabolism in shake flask and spinner flasks culture conditions A) Growth B) Glucose utilization C) Lactate production D) Ammonium production Blue: Shake flask Red: Spinner flask Your local distributor: Eppendorf AG Hamburg Germany eppendorf@eppendorf.com

10 Page 2 (BN 40) JanuarY 2014 A Novel Method for the Expansion of Mesenchymal Stem Cells Using the Eppendorf New Brunswick S41i CO 2 Incubator Shaker due to shear force by the spinner rod. Immunostaining of stem cell surface markers revealed that AdMSCs retained stem cell surface markers during growth under shake flask culture condition (Fig. 2A&B). Adipocyte and osteocyte differentiation assays indicated that most of the AdMSCs from shake flask culture differentiated into either adipocytes or osteocytes successfully (Fig. 3 A&B). A A B CD90 marker (red) Nucleus (blue) Microcarrier bead 200 µm B CD44 marker (green) Nucleus (blue) Microcarrier bead 200 µm Fig. 2: Stem cell marker identification assay for AdMSCs expanded on microcarriers in shake flask A) AdMSCs on microcarrier beads are positive for CD90 stem cell marker (red). B) AdMSCs on microcarrier beads are positive for CD 44 stem cell marker (green). Blue color: nuclear staining by DAPI. Fig. 3: Differentiation assays for AdMSCs expanded on microcarriers in shake flask A) Adipogenic differentiation formed lipid droplets as indicated by Oil red O positive staining. B) Osteogenic differentiation caused calcium mineralization of extracellular matrix as indicated by Alizarin Red S positive staining. Conclusion Stem cell expansion using shake flask conditions appears to be a viable and simple alternative to the spinner flask system. The New Brunswick S41i reduces shearing, eliminates potential cell damage by the spinner rod, decrease the risk of contamination associated with inserting a magnetic stirrer base into the CO 2 incubator and reduces experimental complexity. This method also greatly increases the cell culture capacity whereby a large number of shake flasks can be placed in the New Brunswick S41i simultaneously. In the case of spinner flask culture, a typical incubator without active cooling can only handle the heat emitted from a very limited number of magnetic stirrer bases before causing temperature setpoint overshoot, a significant limitation to the scale-up potential of the spinner method. This reinforces the superiority of the New Brunswick S41i CO 2 Incubator Shaker as an alternative to incubator/spinner based stem cell culture. This method eliminates a scale-up bottleneck while providing highest quality stem cell culture for inoculation of large scale industrial bioreactors for the production of clinical material. The shake flask has lower cost and less parts to disassemble, clean, assemble and autoclave. Literature [1] Kohlstrom et al. (2012): Hybridoma and CHO Cell Culture using the New Brunswick S41i, an Environmentally-Friendly, Low Emission Incubator Shaker. Application Note 255. Available online at applications Readers' service Eppendorf New Brunswick S41i CO2 Incubator Shaker Ref. no. 253 Your local distributor: Eppendorf AG Hamburg Germany eppendorf@eppendorf.com

11 (BN 40) JanuarY 2014 PAGe 3 Quantification of Nucleic Acids Using a Factor-Based Method in the Eppendorf PlateReader AF2200 Tanja Musiol 1, Bettina Scheumer 2, Markus Lapczyna 2 1 Eppendorf AG, Hamburg, Germany 2 Eppendorf Instrumente GmbH, Hamburg, Germany Introduction Determination of concentration via a sample-specific extinction factor is a common approach for the cuvette format, but not for the plate format. The underlying calculation is Lambert- Beer's law: A = ε c d A = Absorbance, ε = Extinction coefficient, c = concentration, d = optical path length For this calculation, knowledge of the sample specific factor, as well as the exact path length are required. In the case of cuvettes, the path length is determined by the shape of the cuvette, as the light beam traverses the cuvette horizontally (Fig. 1). Cuvette Light path Fig. 1: Light beam through the cuvette In the case of absorbance measurements in a plate, the light beam traverses the sample vertically (Fig. 2A). The path length is in this case determined by the filling height. Since the filling height is not only dependent on the volume but also on the geometry of the well, exact determination of the filling height is critical. d In this article it will be shown how, using the Eppendorf Microplate UV-VIS with a defined volume, the concentration of a dsdna solution is determined in the Eppendorf PlateReader AF2200 by means of a factor-based method. Since the exact dimensions of the plate and its wells are known, the software already contains the respective path lengths for pre-defined filling volumes. Calculation of sample concentration is performed analogous to the familiar process employed for instruments using cuvettes. Exact pipetting is of course a prerequisite to ensure uniform filling heights (Fig. 2B). The results from the factor-based method are compared with results evaluated via standard curve and with cuvette measurements carried out in a reference spectrophotometer. Material > > UltraPure Herring Sperm DNA Solution (Life Technologies ; cat. no ) > > Quartz glass cuvette with a 10 mm path length (Hellma Analytics) > > Reference spectrophotometer > > Eppendorf Microplate UV-VIS with UV transparent foil bottom (Eppendorf) > > Eppendorf PlateReader AF2200 (Eppendorf) > > Tris buffer: 0.1 M, ph 8.0 Method The following DNA concentrations are prepared : 5, 10, 25, 50, 60, 80 and 100 ng/µl. Every dilution step is checked in a quartz glass cuvette in a spectrophotometer at a 260 nm wavelength. The dilution steps 5, 25, 50 und 100 ng/µl are used for preparing the standard curve (PlateReader method»uv 260 nm with standards«). The concentrations 80 ng/μl, 60 ng/μl and 10 ng/μl are then determined via the standard curve. Subsequently, all concentrations are again determined using the factorbased method (PlateReader method»uv 260 nm with factor«) and compared to the results obtained with the standard curve, as well as with those obtained from the cuvette measurements on the reference spectrophotometer. All measurements (factor and standard) are performed with 100, 200, 300 µl (one plate for each volume). During the course of factor-based measurements, absorbance values at the following wavelengths are determined: 260 nm, 280 nm and 340 nm. The value generated at 340 nm is taken into consideration as a reference value in order to minimize the influence of contamination. The value obtained at 280 nm is used to calculate the A260/ A280 ratio and to determine the purity of the DNA. Pure DNA shows a ratio between 1.8 and 2.0. Lower ratios may indicate protein contamination. The absorbance measurement at 260 nm is used to calculate the DNA concentration following Lambert-Beer's law. Calculation of concentration is performed by the instrument's software by using the sample specific extinction factor of 50 for double-stranded DNA (dsdna). A Microplate B Microplate d Light path d Light path Volume Volume Fig. 2: Photometric determination in a microplate. The path length is determined by the filling height. A) Different liquid levels due to unreproducible pipetting or high variances in the well geometry B) Photometric determination in plate readers requires identical liquid levels. Your local distributor: Eppendorf AG Hamburg Germany eppendorf@eppendorf.com

12 Page 4 (BN 40) JanuarY 2014 Quantification of Nucleic Acids Using a Factor-Based Method in the Eppendorf PlateReader AF2200 Absorbance y = x R²= Concentration [µg/ml] Fig. 4: 100 µl measurement; standard curve Fig. 3: Screenshot of the method»nucleic Acid quantification (UV 260 nm with factor)«from the software of the PlateReader AF2200. (Example: measurement of 100 µl). This factor states that an absorbance value of 1, measured in a 10 mm light path, translates to a DNA concentration of 50 μg/μl [1]. Results Figures 4, 5 and 6 show the respective standard curve obtained by 100, 200 and 300 µl measurement. All standard curves show optimal linearity. Table 1 gives an overview of the factor-based and standard curve based measurements. In addition the corresponding results from the cuvette measurements with the reference spectrophotometer are displayed. Same or even better are the results obtained with the factorbased measurements compared to measurements with standard in relation to the reference measurements performed in cuvettes. Conclusion As clearly shown by the comparative analyses of measurement results, factorbased determination of sample concentrations is possible in the Eppendorf PlateReader AF2200 when using the Eppendorf Microplate UV-VIS. The results are comparable amongst each other, as well as against reference measurements in standard cuvettes. The same is true for the comparison of factor-based results with analyses via standard curves, which represent the common method for concentration determination in a plate reader. Since filling height determines the path length, which is a fundamental parameter of concentration determination, highly reproducible dispensing of sample volumes is critical, as well as the use of plates with known and consistent well geometry. Absorbance Absorbance Concentration [µg/ml] Fig. 5: 200 µl measurement; standard curve Splashes on the rim of the well or the formation of a pronounced meniscus (e. g. resulting from detergent containing buffers) are to be avoided. Taken together, it is safe to state that factorbased calculation of the concentration in the Eppendorf PlateReader AF2200, in combination with the Eppendorf Microplate UV-VIS, represents a qualitative substitute to the common method of extrapolation from a standard curve. By eliminating the effort required to generate a standard curve, this method is very economical compared to the current common procedures, since both time and money are saved. Literature y = x R²= y = x R²= Concentration [µg/ml] Fig. 6: 300 µl measurement; standard curve Prepared dsdna concentration in [µg/ml] Cuvettes based measurements performed in a spectrophotometer (references) [µg/ml] Filling volume in the plate [µl] Results of standard curve based measurements performed in the Microplate UV-VIS [µg/ml] Results of factor-based measurements performed in the Microplate UV-VIS [µg/ml] Variance between standard curve and cuvettes based measurements [%] Variance between factor and cuvettes based measurements [%] Variance between factor and standard curve based measurements [%] Table 1: Summary of factor-based and standard curve based measurements of dsdna performed in the Eppendorf PlateReader AF2200 using the Eppendorf Microplate UV-VIS, compared to measurements performed in cuvettes using a spectrophotometer [1] Gallagher, Sean R. (2001), Quantification of DNA and RNA with Absorption and Fluorescence Spectroscopy, Current Protocols in Cell Biology, Appendix 3D Readers' service Eppendorf PlateReader AF2200 Ref. no. 261 Your local distributor: Eppendorf AG Hamburg Germany eppendorf@eppendorf.com

13 (BN 40) JanuarY 2014 PAGe 5 Comparison of Eppendorf Tubes 5.0 ml to Conical 15 ml Tubes in Regard to Releasable UV-Absorbing Substances (Leachables) Natascha WeiSS 1, Frauke Gotzhein 2 1 Eppendorf AG, Hamburg, Germany 2 University of Hamburg, Academic Program Molecular Life Sciences, Hamburg, Germany Abstract Substances which are released from plastic consumables (»leachables«) may have an influence on laboratory experiments. If they absorb light in the UV range, they can easily be detected by heating water in reaction vessels, followed by photometric analysis. Such substances, for example, can easily falsify DNA measurements. The present Application Note describes experimental comparison of Eppendorf Tubes 5.0 ml and conical 15 ml vessels according to this method. It can be shown that a significant amount of leachables is released from the different 15 ml vessels. In contrast, for Eppendorf Tubes (and a glass control vessel) the extinction is so low that photometric detection methods are not compromised. Introduction The topic of»leachables«(substances leaching from plastics) has been in the news for some years, and it is especially significant in the fields of food packaging and beverage containers [1, 2]. thus obtained from Eppendorf reaction vessels did not have significant adverse effects on photometric analyses. The experiments in the present Application Note were conducted in accordance with the test described above [7, 8]. The aim of this study was the comparison of the Eppendorf Tubes 5.0 ml with conical 15 ml vessels from different manufacturers with regards to leaching of UV-absorbing substances. Materials and methods Three Eppendorf Tubes 5.0 ml and three conical 15 ml vessels each of four different manufacturers (B, C, F, V) were filled with 1 ml water (for molecular biology) and incubated for 30 min at 90 C* in the Eppendorf ThermoMixer comfort, while mixing occurred at 600 rpm. Similar conditions are found in many protocols for sample preparation, such as sample lysis. Following incubation, samples were allowed to cool down for 10 min. The samples were scanned across the range between 220 nm to 340 nm in UVettes, using the Eppendorf BioSpectrometer. The averages and standard deviations were determined from the three respective replicates. The extinctions may be considered proportional to the amount of substances leaching from the vessels. Non-incubated water was used as the blank, while water which had been incubated in a glass container served as the control. The dsdna concentration which could theoretically be derived from the measured values was calculated from the extinction at 260 nm using the factor 50 μg/ml per unit of extinction. Results and discussion Fig. 1 shows the absorbance spectra of water obtained from the different vessels in which it had been incubated for 30 min at 90 C while being mixed at 600 rpm. In addition, the insert lists the dsdna concentrations theoretically derived from the extinctions measured at 260 nm, which may therefore be misinterpreted as DNA. A glass control was used since no significant leaching of UV-absorbing material is observed under the given experimental conditions [7]. In 2008 a publication in Science brought to light the problems associated with plastic consumables in laboratories. In this and other publications the influence of different additives which may leach from plastic containers and pipette tips on enzyme activity was demonstrated by means of specific enzyme assays [3, 4, 5, 6]. Even standard laboratory methods such as photometric detection of nucleic acids and proteins can be falsified by leachables. Using a simple test system which includes heating of water in reaction vessels (with standard laboratory methods), followed by an absorbance scan in the UV range, it could be demonstrated that UV-absorbing substances leach from single use products [7]. In this publication, as well as in the Eppendorf Application Note 235 [8] it was further shown that water samples Extinction [OD] Absorbance scan of water following incubation at 90 C Theoretical dsdna concentrations Manufacturer F: 5.01 µg/ml Manufacturer C: 4.13 µg/ml Manufacturer B: 3.37 µg/ml Manufacturer V: 2.31 µg/ml Eppendorf: 0.81 µg/ml Glass value: 0.35 µg/ml Wavelength λ [nm] Manufacturer C Manufacturer B Manufacturer F Manufacturer V Eppendorf Glass value Fig. 1: Absorbance spectra of water following incubation in Eppendorf Tubes 5.0 ml and conical 15 ml vessels by other manufacturers for 30 min at 90 C Your local distributor: Eppendorf AG Hamburg Germany eppendorf@eppendorf.com

14 Page 6 (BN 40) JanuarY 2014 Comparison of Eppendorf Tubes 5.0 ml to Conical 15 ml Tubes in Regard to Releasable UV-Absorbing Substances (Leachables) Extinction [OD] Absorbance spectra of water with standard deviation Wavelength λ [nm] Manufacturer C Manufacturer B Manufacturer F Manufacturer V Eppendorf Glass value Fig. 2: Absorbance spectra of water with standard deviations in the range between 230 nm and 280 nm As expected, the glass vessel produces the lowest values. The absorbance spectrum of water obtained from Eppendorf vessels is only slightly higher than that of the control. The extinction value at 260 nm translates to a theoretical dsdna concentration of 0.35 μg/ml for the glass control, and to 0.81 μg/ml for the Eppendorf Tubes. These values are below the recommended limit of quantification for the BioSpectrometer (sample extinction values of or dsdna concentration of μg/ml, respectively [9]). Since other influencing factors such as particles, turbidity or bubbles inside the solution significantly compromise the results at very low extinctions, these data are considered not meaningful with respect to the leaching of substances. In contrast, significant absorbance spectra are recognizable in all water samples which were heated in the conical 15 ml tubes. The absorbance values obtained at 260 nm translate to theoretical dsdna concentrations of 2.31 μg/ml to 5.01 μg/ml. It had been shown previously that standard laboratory methods during which sample temperature is elevated (incubation, PCR, centrifugation, sonication) lead to leaching of UV-absorbing substances from the containers [7, 8]. This may yield falsely elevated results during photometric analyses of molecules such as nucleic acids and proteins which are primarily conducted at 260 nm 280 nm. In the case of samples of low concentration, these errors may compromise subsequent experiments. Additional problems may be presented by the variability of these errors. Fig. 2 shows an excerpt of the range between 230 nm and 280 nm of the same absorbance scan, with the standard deviations overlaid. The values obtained with water from the 15 ml conical vessels show considerable variation, indicating that the substances contained in the vessel material leach at different rates. Thus, the error is not a constant factor but is also subject to large variation. Conclusion The data presented in this Application Note show that under the influence of heat, UV-absorbing substances leach from the tested conical 15 ml vessels. These leachables can compromise or falsify downstream analyses such as DNA quantification. The new Eppendorf Tube 5.0 ml yields an extinction which is only slightly above that of the control and thus represents a very good alternative for a large volume tube when minimizing the influence of leachables in sensitive methods is crucial. Owing to the certified omission of problematic additives such as lubricants, biocides or plasticizers in the raw material as well as during the production process, these vessels are very well suited for sensitive analyses. This advantage was previously also demonstrated for smaller tube formats provided by Eppendorf [6, 7, 8]. *Temperature range of Eppendorf Tubes 5.0 ml is 86 C to 80 C. For incubation at 90 C the Tube Clip 5.0 ml must be used to prevent tubes from opening. Literature [1] Vandenberg LN, Maffini MV, Sonnenschein C, Rubin BS, Soto AM. Bisphenol-A and the great divide: a review of controversies in the field of endocrine disruption. Endocr Rev 2009; 30: [2] Shotyk W, Krachler M, Chen B. Contamination of Canadian and European bottled waters with antimony from PET containers. J Environ Monit 2006; 8: [3] 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(5903):917. [4] Watson J, Greenough EB, Lett JE, Ford MJ, Drexler DM, Belcastro JV, Herbst JJ, Chatterjee M, Banks M. Extraction, identification, and functional characterization of a bioactive substance from automated compound-handling plastic tips. J Biomol Screen 2009; 14(5): [5] McDonald GR, Kozuska JL, Holt A. Bioactive Leachates from Lab Plastics. G.I.T. Laboratory Journal Europe 2009; 13: [6] Olivieri A, Degenhardt OS, McDonald GR, Narang D, Paulsen IM, Kozuska JL, Holt A, On the disruption of biochemical and biological assays by chemicals leaching from disposable laboratory plasticware. Can J Physiol Pharmacol 2012; 90(6): [7] Lewis LK, Robson M, Vecherkina Y, Ji C, Beall G. Interference with spectrophotometric analysis of nucleic acids and proteins by leaching of chemicals from plastic tubes. BioTechniques 2010; 48(4) [8] Application Note 235: The influence of UVabsorbing substances released from plastic containers (leachables) on photometric analyses ( [9] Operating manual Eppendorf BioSpectrometer ( Readers' service Eppendorf Tubes 5.0 ml Ref. no. 264 Your local distributor: Eppendorf AG Hamburg Germany eppendorf@eppendorf.com

15 (BN 40) JanuarY 2014 PAGe 7 Comparative Run Time Evaluations of PCR Thermal Cyclers Nils Gerke, Eppendorf AG, Hamburg, Germany Abstract For the purpose of comparing the speed of different thermal cyclers, the isolated consideration of heating and cooling ramp rates cited in the technical specifications often does not reflect the actual run times. An estimate of actual run times based on these technical ramp rates may thus lead to false conclusions. Whereas the Mastercycler pro S and the Mastercycler nexus GSX1, as expected from their fast ramp rates, achieved the shortest total PCR run times in these evaluations, some thermal cyclers made by other manufacturers showed noticeably slower run times despite similar cited ramp rates. Introduction Besides control accuracy and temperature homogeneity, the customary technical details of a thermal cycler also include the ramp rate of the thermoblock. The ramp rate, in particular, is not subject to a uniform standard; instead, manufacturers state a variety of parameters, such as: > > maximum heating and cooling rate > > maximum ramp rate > > average ramp rate > > maximum sample ramp rate. Thus, the user is left with the option of estimating the actual ramp rates based on these diverse statements. Therefore, comparative investigations were undertaken in order to evaluate whether the details pertaining to the ramp rates stated in the technical specifications are suitable for estimating the total run times of PCR applications on thermal cyclers. Materials and methods 48 positions of a 96-well plate (Eppendorf twin.tec PCR Plate 96, low profile) were filled with 25 μl water, respectively (Fig. 1). Fig. 1: Positions indicated in black of the 96-well PCR plate were filled with 25 μl water each. The plate was subsequently sealed with the Heat Sealing PCR Film (Eppendorf), centrifuged for 1 min at g, placed into the thermal cycler and subjected to a standard 3-step PCR program (Fig. 2). A Fig. 2: 3-step PCR program for run time determination A) Start of run time measurement B) End of run time measurement The run times were determined for the Mastercycler pro S, Mastercycler nexus GSX1, Mastercycler nexus gradient and Mastercycler nexus, and eight competing thermal cyclers*. b In cases where the respective thermal cycler software allowed for different temperature control modes or reaction volume settings, the fastest ramping speed and/or the lowest volume setting were chosen. Measurement of total run time was initiated immediately following commencement of the first temperature step, and it ended immediately after the temperature of the final step had been reached. For thermal cyclers which record and save a detailed run protocol, it was possible to determine the total run time following completion of the run by consulting the records. Apart from the Mastercycler pro and Mastercycler nexus models, whose run protocols may be exported as pdf files for documentation purposes subsequent to the run (Fig. 3), a detailed, exportable record could be obtained from only three of the competing thermal cyclers. Results and discussion The evaluation of the ramp rates stated by the manufacturers, in comparison with the empirically determined run times, highlighted the fact that isolated consideration of ramp rates in accordance with technical data is not suitable for the reliable prediction of the actual run time of a PCR program (Table 1). Fig. 3: Screenshot from a Mastercycler pro run protocol, exported as pdf file, from the instrument software (additional information, e. g. user, program details and additional cycler settings, is not displayed in this section). Your local distributor: Eppendorf AG Hamburg Germany eppendorf@eppendorf.com

16 Page 8 (BN 40) JanuarY 2014 Comparative Run Time Evaluations of PCR Thermal Cyclers > > Temperature control modes or reaction volume settings may also exert considerable influence on ramping behavior [1]. This may even lead to the need to re-optimize a reaction following the transfer of a PCR system from one thermal cycler to another [2]. Conclusion Fig. 4: Mastercycler pro S with silver block On the one hand, the thermal cyclers Mastercycler pro S (Fig. 4) and the Mastercycler nexus GSX1 (Fig. 5) showed, as expected, the shortest total PCR run times, in accordance with the fast ramp rates cited. On the other hand, the run times of certain competing thermal cyclers were considerably longer than would be expected from the ramp rates stated in the manufacturers' technical specifications. It is thus evident that the thermal cyclers V, T and R were considerably slower in their actual PCR run times than the Mastercycler nexus gradient and the Thermal cycler Run time (hh:mm:ss) Fig. 5: Mastercycler nexus GSX1 with silver block Mastercycler nexus, despite the fact that the respective manufacturers had cited faster ramp rates for these thermal cyclers in their technical specifications. It can be assumed that the following parameters contribute strongly to the observed discrepancies: > > For the different thermal cyclers the maximum ramp rates stated in the technical manuals are reached for different periods of time during the ramping process from one temperature to the next possibly for only a short time during each ramping phase for certain thermal cyclers. Ramp rate according to technical data ( C/s) Mastercycler pro S 00:40:12 6 Mastercycler nexus GSX1 00:42:31 5 C 00:46:50 5 These comparative investigations have shown that the isolated consideration of ramp rates often bears limited meaningfulness and may even lead to false conclusions with regards to the estimation of the actual PCR run time of a given thermal cycler. For accurate evaluation of the ramping performance of a thermal cycler it is imperative that the manufacturer makes the information on all relevant parameters, e. g. detailed description of selectable temperature control modes, available to the user. Besides consideration of the technical data, for the purpose of an all-encompassing assessment of the performance of a thermal cycler it is strongly recommended to test the instrument in a demo-setting with regards to hardware, software and PCR applications. *Tests were performed on one cycler each of every model type. Literature [1] Application Note [2] Hughes S., Moody A. (eds.): PCR. Scion Publishing Limited; P 00:48:58 5 S 00:50:31 6 Mastercycler nexus gradient 00:51:26 3 Mastercycler nexus 00:51:53 3 V 00:52:22 5 T 00:53:20 4 R 00:56:27 5 G 00:56: A 01:03:13 3 Table 1: Total run time of a standard 3-step PCR protocol using the fastest settings possible in the instrument software. Due to diverse manufacturers' statements of ramp rates, only the maximum ramp rate which could be found according to the technical data for an instrument is presented here. Readers' service Mastercycler Family Ref. no. 265 Your local distributor: Eppendorf AG Hamburg Germany eppendorf@eppendorf.com

17 Best Conditions for Your PCR News 9 Kay Körner, Eppendorf AG Best Conditions for Your PCR The Mastercycler nexus family by Eppendorf provides you with a wide selection of different PCR cyclers. Whether you choose the silver block, the flat block or the universal block, with or without gradient function; whether a single unit or a network of up to three units with Eppendorf you will find the ideal configuration which meets the demands of your laboratory on an efficient PCR cycler. This feature allows a number of different assays to be run on the same instrument, which is of advantage to larger research groups using different formats of consumables. Regardless of which block you will choose, all instruments of the Mastercycler nexus family offer numerous advantages: > > Small footprint Silver block (heating rate approx. 5 C/s) Silver block: for higher heating and cooling rates The new Mastercycler nexus X1 combines the modern and intuitive software of the Mastercycler nexus with a fast 96-well silver block for higher heating and cooling rates. The Mastercycler nexus X1 is fast and user-friendly. It requires little space and little electricity, and it sends you an when it is finished. What more could one ask of a PCR cycler?»to raise new questions, new possibilities, to regard old problems from a new angle, requires creative imagination and marks real advance in science.«albert Einstein Mastercycler nexus with silver block: fast, intuitive, reliable Flat block: the solution for unusual problems Special applications often require special consumables. The Mastercycler nexus flat, with its flat block without wells, offers the ideal base for slides and other unusual formats of consumables. During in situ PCR the results are typically influenced by the characteristic heat conductivity of the respective in situ adapter. Thanks to the flat block, the Mastercycler nexus flat is capable of heating or cooling your slides directly, thus foregoing the need for an adapter. Universal block for maximum flexibility The Mastercycler nexus with universal block can accommodate 96-well PCR plates, 0.2 ml PCR tubes, 0.2 ml PCR strips, as well as 0.5 ml PCR tubes. > > Intuitive graphic programming > > Up to two instruments may be connected to a central instrument > > Optional gradient > > notification > > flexlid concept: automatic height adjustment of the lid to accommodate a variety of consumables > > 2 year warranty > > Optional self-test function > > Low noise emission > > Low power consumption Further information Additional details are available at or in the current brochure which may be ordered using the reference number denoted below. Of course we are always happy to offer in-person consultations. Mastercycler Family Ref. no. 265

18 10 Straight from the lab Ergonomics in Today's Laboratory Routine Sabine Kühn, Eppendorf AG Ergonomics in Today's Laboratory Routine Healthy work conditions are gaining in importance, not the least due to demographic developments in industrialized nations. Many studies show that laboratory work in particular harbors numerous health risks if work conditions fail to fulfill a holistic ergonomic approach. According to the definition by the International Ergonomics Association IEA, the federation of ergonomics and human factors societies around the world, a holistic, ergonomic approach encompasses the consideration of physical, cognitive and organizational ergonomics. This is where the Eppendorf PhysioCare Concept comes in an inclusive, ergonomic product concept which integrates the three areas mentioned above in a logical and comprehensible fashion. In order to design the workplace/laboratory under ergonomic considerations, the PhysioCare Concept differentiates between three core areas also called»spheres«. Sphere 1: The user Sphere 1 focuses on the users' needs with respect to ergonomic product design as well as product performance optimized to their individual needs. It is important to Eppendorf that all products, in addition to exceptional ergonomic design, ensure simple and intuitive handling, and that product performance thus satisfies the highest demands. ventilation so that neighboring work spaces are not impacted. At the same time, a well thought-out concept for product accessories contributes to the facilitation of optimized work space organization. Sphere 3: The workflow Sphere 3 attends to the actual work flow. The aim is optimization of laboratory processes, with the final goal of improving results for the entire organization. To this end, Eppendorf supports its users with a comprehensive selection of consulting and services, as well as with education through the Eppendorf Training Center. Further information Comprehensive information about the PhysioCare Concept as well as tips and ideas for optimizing your laboratory routine are available at News Correct Pipetting Pipetting is one of the most important steps towards a correct analysis result. However, pipetting technique or maintenance and care of pipettes are seldom taught in schools or universities. Cross your heart: Do you, for instance, know the difference between a direct displacement pipette and an air cushion pipette? What are the optimum areas of use for both models? When is the technique of reverse pipetting useful? How are systematic and random errors calculated? What is the connection between ergonomic working conditions and user health? Our experience, your advantage We at Eppendorf know that correct pipetting is no coincidence but rather a precise craft. This knowledge, along with the experience gathered over more than 50 years of liquid handling, is passed on by the specialists at the Eppendorf Training Center (ETC) in our popular customer seminars. These training sessions are conducted in small groups, optionally in Eppendorf training rooms, or directly on-site at your facility, by our experienced and certified trainers. Further information and local offers are available at epservices.* We are also pleased to offer you a personal consultation. Sphere 2: The laboratory Sphere 2 regards the system»laboratory«with respect to harmonic integration of products in line with the specific demands of the workplace. The resulting challenge for Eppendorf is, for example, to design instruments with as little noise emission as possible or to place instrument The Eppendorf PhysioCare Concept integrates ergonomics holistically into the modern laboratory routine. * The Eppendorf Training Center offers are available in selected countries only and courses may vary.

19 »Smooth Operators«: New Micromanipulators Innovation 11 Heide Niesalla, Eppendorf AG»Smooth Operators«: New Micromanipulators For the past 25 years, Eppendorf micromanipulators have been synonymous with highly precise, innovative systems which are used around the world for a large number of applications. Precision, fast and easy sample processing, as well as»real time feeling«, were decisive criteria during the development of the new and innovative micromanipulators TransferMan 4m and TransferMan 4r. The exceptionally direct transmission of movement in all directions makes users feel as though they are working with a mechanical system. This feature was especially verified by clients who tested the new instruments in direct comparison with mechanic-hydraulic competitors' systems. The new Eppendorf DualSpeed joystick expands the precise and intuitive direct movement control by an additional dynamic speed mode in order to cover longer distances or to accelerate sample processing. The newly introduced electronic coupling with the Eppendorf PiezoXpert enables semi-automatic piezo-assisted cell penetration. This is of interest, for example, for the successful injection of plant cells where, on account of the thickness of the cell wall, larger distances need to be bridged. Further information Detailed Application Notes and additional information about the topic are available at The new TransferMan systems combine intuitive handling with outstanding precision. Thoroughly tested by clients During the development phase of the TransferMan 4m and 4r we set great store by our clients' feedback: in particular robustness and the new product features were evaluated critically during intensive application tests, which were conducted within routine applications. and approved! The new TransferMan systems combine intuitive handling with outstanding precision. Simplified workflow Eppendorf micromanipulators are used, among other applications, for artificial insemination, generation of genetically modified animals and plants, as well as for selection and transfer of single cells, micro-particles and crystals. Thanks to pre-defined, application-specific user profiles such as»cell transfer«and»dna injection«, the multifunctional instruments simplify individual work processes. Intelligent additional functions such as saving of positions are easily accessed within the individual profiles. Flexible application Thanks to the flexible assembly and installation concept, installation of the new manipulators on all common microscopes is possible. Semi-automatic injections are enabled by combination with the electronic Eppendorf microinjectors. Tip: Watch the»new ones«in action on the Eppendorf YouTube channel! TransferMan 4m/TransferMan 4r Ref. no. 266

20 12 News New 1 Liter Vessels Expand the BioBLU Family Claudia Huether-Franken, Eppendorf AG, Bioprocess Center Europe, Juelich, Germany New 1 Liter Vessels Expand the BioBLU Family With the new single-use bioreactors BioBLU 1c for cell culture and BioBLU 1f for microbial applications, Eppendorf is expanding the BioBLU family of stirred-tank rigid-wall single-use bioreactors. The new 1 L vessels close the gap between the BioBLU 0.3c/f single-use mini bioreactors and the larger vessels of the BioBLU family. For users in cell culture who would like to combine the advantages of single-use technology with the proven qualities of stirred-tank bioreactor design, a uniquely comprehensive portfolio of stirred rigidwall single-use bioreactors with working volumes from 100 ml to 40 L is now available. Efficient through parallel processing The BioBLU 1c/f single-use bioreactors were developed specifically for use with DASGIP Parallel Bioreactor Systems BioBLU 1c for cell culture BioBLU 1f for microbiology which can operate 4, 8 and more bioreactors simultaneously. Users benefit from parallelizing their processes for cultivation of animal and human cells as well as for microbial applications with bacteria and yeast. Reliable and comparable results based on precise process control, and time savings by combining parallel workflows, paired with the minimal setup times required for single-use bioreactors sustainably accelerate the development of bioprocessing. Effective process planning and automation, design of experiment (DOE) and comprehensive bioprocess information management additionally maximize time and cost effectiveness of the process. Established design combined with single-use technology The fully instrumented BioBLU 1c/f rigid-wall single-use bio - reactors can be operated with working volumes between 320 ml and 1.25 L for the cell culture variant, and 250 ml to 1.25 L for microbial applications. All critical process parameters such as temperature, ph and soluble oxygen can be monitored and controlled with industry standard sensors. Integrated dip tubes allow controlled addition of liquids and sampling as well as submersed or headspace-gassing. The BioBLU 1c comes with two 3 x 45 pitched blade impellers, the BioBLU 1f with three 6-blade Rushton-type impellers. The specially developed, encapsulated and magnet-coupled stirrer drive enables secure and sterile agitation with up to 600 rpm in cell culture and 1,600 rpm for microbial applications. Mass transfer rates that meet the high demands of microbial applications are guaranteed. The innovative liquid-free Peltier condenser ensures optimal cool down of exhaust. In addition, the BioBLU 1f is equipped with four innovative cooling baffles which support precise temperature control even under conditions of strong biological heat production such as that occurring with modern high cell density processes. All wetted materials of the BioBLU 1c/f single-use bioreactors are certified»usp Class VI«and are in compliance with US-FDA requirements. More information at

21 A Really Cool Product Family: New Brunswick ULT Freezers Straight from the lab 13 Mary Lisa Sassano, Eppendorf, Inc., Enfield, CT, USA A Really Cool Product Family: New Brunswick ULT Freezers The current Eppendorf line of New Brunswick Ultra-Low Temperature (ULT) freezers has become so comprehensive and versatile that virtually all users will easily find a freezer that fits the requirements of their labs. Every instrument is individually tested and performance-certified. Offered in both upright and chest styles, New Brunswick ULT freezers comprise 15 different models with L capacities. Impetus to energy-efficiency New Brunswick ULT freezers are wellknown for their energy-efficient performance. Innova freezers provide up to 30 % more storage capacity compared to traditionally insulated freezers by using advanced vacuum insulated panel technology which allows a reduction in freezer wall thickness.»premium«freezers offer the same top-quality, but use conventional foam insulation for a cost-saving alternative. High Efficiency HEF and Green»G«freezers further reduce energy consumption, making them among the most energy-efficient in the market. HEF freezers, for example, reduce energy consumption by up to 59 % when compared to competitive models. Vacuum insulation panels combined with traditional polyurethane foam form a 130 mm thick layer of insulation. These freezers, in addition, feature a powerful, low-noise condenser fan and compressor system thus providing exceptional energy efficiency. The 50 Hz versions of HEF and Green models additionally use environmentally-safe hydrocarbon-based refrigerants to further reduce energy consumption and greenhouse gases. Smart features All models include ergonomically positioned easy-open doors with a molded handle, adjustable height shelves, low noise and low heat output. A bright LED control panel mounted at eye level on uprights is easy-to-read, and flush mounted for easy wipe-down. A washable filter is front mounted and easily accessed without tools. Also, a heated air vent with ice-cleaning plunger prevents vacuum formation, allowing the door to be quickly opened throughout the day. These ULT freezers are designed to fit through standard doorways and elevators, allowing for ease of movement. Tip: The TCA-3 Temperature Monitoring System offers added security. This optional unit combines an independent temperature monitor with an alarm, electronic chart recorder, and an autodialer into one small pod. The TCA-3 provides users a considerable measure of added security for their sample protection. < 80 mm > < 130 mm > < 130 mm > Innova walls Premium walls HEF walls Innova Freezers reduce insulating wall thickness to provide up to 30 % more storage capacity HEF Freezers improve energy efficiency by adding vacuum insulation panels to traditional foam in a 130 mm space Polyurethane Foam Vacuum Insulation Panels There is a New Brunswick freezer perfectly suited for every lab! New Brunswick HEF Freezers Ref. no. 267

22 14 Service Prize Competition Prize Competition The solution of the prize competition of BioNews No. 38 was»combitips advanced«. Yuki Sugeno (G and G science Company, Fukusimaken, Japan) won the first prize. Have fun in our new crossword! How to find out the solution: Simply arrange the letters in the light grey boxes of the crossword in the correct order. Send us the solution until 30 th June You can either use the reply fax (p. 15), send us an to bionews@eppendorf.de, or participate online at All correct answers will be considered for a prize. Winners will be notified in writing. Cash payment of the prize is not possible. No recourse to legal action. The judges' decision is final. Eppendorf employees and their families may not participate. The winner of the first prize will be published in BioNews No st Prize: 1 Multipette M4* with 2 sets of Combitips advanced of your choice *(US/CAN: Repeater M4) 2 nd to 5 th Prize: 1 Amazon Voucher worth 50. Euros 6 th to 15 th Prize: 200 bonus ep-points each 48 ACROSS 1 Centrigrade temperature scale 7 These are a-changin (according to Bob D.) 12 It's fun to stay there 13 Opposite of yes 14 Computer-aided design (abbrev.) 15 Popular lab instrument 17 Chemical symbol for lithium 18 Human immunodeficiency virus (abbrev.) 19 Word expressing approval or assent (abbrev.) 20 Spanish definite article 21 ISO code of Estonia 22 Warning signal 24 Ribonucleic acid (abbrev.) 26 Professional Golfers Association (abbrev.) 27 Male given name 28 ISO code of Turkey 29 ISO code of Nauru 30 Not fake or false (e.g., in Madrid) 31 ISO code of Greenland 33 The B in BMI 35 Capital of Norway 38 Number prefix denoting two 40 Moon of saturn 41 Greek prefix for young, new 42 Object detection system based on radio waves 45 Founder of an annual international awards bestowed in a number of categories (first name) 46 Chemical symbol for silicon 48 Biotechnological process DOWN 1 Device used for PCR 2 Electronic message 3 Flat panel display (abbrev.) 4 ISO code of Saudi-Arabia 5 Comprehensive broad and versatile 6 Morse code distress signal 7 Travel by walking 8 Male given name 9 Part of the name of Eppendorf thermocyclers 10 A particular part of something 11 Severe acute respiratory syndrome (abbrev.) 15 Princess of Wales (short name) 16 Flows through Turin and Piacenza 18 Female given Name 22 Chemical symbol for silver 23 To sell or write 25 Noble gas 26 Thin, long instrument used for exploration 29 nota bene (abbrev.) 32 French law 34 Electronic component 36 Completes Lucia, Louis and Tropez 37 Tool of the American cowboy 39 Rail vehicle (e. g., in Vienna, Austria) m (abbrev.) 43 Typical British drink (produced using 48 across) 44 The A in MOMA 47 Chemical symbol for indium Disclaimer and Trademarks Information Amazon is a registered trademark of Amazon.com, Inc., USA. DASGIP is a registered trademark of DASGIP Information and Process Technology GmbH, Germany. HEF and Innova are registered trademarks of New Brunswick Scientific Co. Inc, USA. Hellma is a registered trademark of Hellma GmbH & Co. KG, Germany. Life Technologies is a registered trademark of Life Technologies Corp., USA. Science is a registered trademark of American Association for the Advancement of Science, USA. UltraPure is a trademark of Life Technologies Corp., USA. YouTube is a registered trademark of Google Inc. Corp., USA. Eppendorf, the Eppendorf logo, BioBlu, Combitips advanced, ep Dualfilter T.I.P.S., epmotion, Eppendorf Biopur, Eppendorf BioSpectrometer, Eppendorf LoBind, Eppendorf PhysioCare Concept, Eppendorf PiezoXpert, Eppendorf Reference, Eppendorf Thermomixer, Eppendorf ThermoMixer, Eppendorf Tubes, Eppendorf twin.tec, ep-points, epservices for premium performance, ept.i.p.s., flexlid, g-safe, Mastercycler, Multipette, PhysioCare Concept, Repeater, Thermomixer, TransferMan, UVette are registered trademarks of Eppendorf AG, Germany. Eppendorf DualSpeed, Eppendorf Quality, Eppendorf ThermoStat, the epservices logo, New Brunswick and the New Brunswick logo are trademarks of Eppendorf AG, Germany. Errors and omissions excepted. Copyright January 2014.

23 Reply Fax / Readers' Service Service 15 To Eppendorf AG, Hamburg, Germany, Attn. Carolyn Taubert, Fax (+49) (Please print or type, no private address) Company / University / Clinic Title / Name Institute Address Department / Function Address Telephone Please send me information on the following products: Kindly help us to update your subscription details: 253 New Brunswick S41i CO 2 Incubator Shaker Please change my details (see above). 261 Eppendorf PlateReader AF2200 I am a new subscriber! Please send me Eppendorf BioNews on a regular basis 262 ep Dualfilter T.I.P.S. SealMax (2 issues per year, free of charge). 264 Eppendorf Tubes 5.0 ml I have comments on BioNews No. 40: 265 Mastercycler Family 266 TransferMan 4m/r 267 New Brunswick HEF Freezers 268 Multipette M4 (US/CAN: Repeater M4) 269 Eppendorf Reference 2 Eppendorf General Catalog 2014 Visit to subscribe or to request literature online! Solution of BioNews No. 40 prize competition: F E E E T Closing date: 30 th June 2014 Visit to participate online.

24 Careers ABN There s only one Galileo Galilei B orn in 1564, Galileo Galilei once contemplated a career in the priesthood. It s perhaps fortunate for science that upon the urging of his father, he instead decided to enroll at the University of Pisa. His career in science began with medicine and from there he subsequently went on to become a philosopher, physicist, mathematician, and astronomer, for which he is perhaps best known. His astronomical observations and subsequent improvements to telescopes built his reputation as a leading scientist of his time, but also led him to probe subject matter counter to prevailing dogma. His expressed views on the Earth s movement around the sun caused him to be declared suspect of heresy, which for some time led to a ban on the reprinting of his works. Galileo s career changed science for all of us and he was without doubt a leading light in the scientific revolution, which is perhaps why Albert Einstein called him the father of modern science. Want to challenge the status quo and make the Earth move? At Science we are here to help you in your own scientific career with expert career advice, forums, job postings, and more all for free. For your career in science, there s only one Science. Visit ScienceCareers.org today. For your career in science, there s only one ScienceCareers.org Career advice I Job postings I Job Alerts I Career Forum I Crafting resumes/cvs I Preparing for interviews