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No. 40 – 2014

(BN 40) JANUARY 2014 PAGE3

Yourlocaldistributor: www.eppendorf.com/contact EppendorfAG · 22331 Hamburg · Germany· E-mail: [email protected] · www.eppendorf.com

QuantificationofNucleicAcidsUsing aFactor-BasedMethod

inthe EppendorfPlateReaderAF2200

TANJAMUSIOL1,BETTINASCHEUMER2,MARKUSLAPCZYNA2

1EPPENDORFAG,HAMBURG,GERMANY2EPPENDORFINSTRUMENTEGMBH,HAMBURG,GERMANY

Introduction

Determinationofconcentrationvia a

sample-specificextinctionfactor is acommonapproachfor the cuvette

format, but not for the plate format.

TheunderlyingcalculationisLambert-

Beer's law:

For this calculation, knowledge ofthe

sample specificfactor, as well as theexact pathlengthare required. Inthe

case ofcuvettes, the pathlength isdeterminedbythe shapeofthe cuvette,

as the light beam traverses the cuvettehorizontally(Fig. 1).

Inthecase ofabsorbancemeasurements

ina plate, the light beam traverses thesample vertically(Fig. 2A).

The pathlengthis inthis case deter-

mined bythe filling height. Since thefilling height is not onlydependent on

the volume but alsoon the geometryofthe well, exact determinationofthe

filling height is critical.

Inthis article it will be shownhow,

usingtheEppendorf MicroplateUV-VISwithadefinedvolume,the concentration

ofa dsDNAsolution is determined inthe EppendorfPlateReader AF2200 by

means ofa factor-based method. Sincethe exact dimensions ofthe plate and

its wells are known, the softwarealreadycontains the respective path

lengthsforpre-defined fillingvolumes.Calculationofsampleconcentrationisperformed analogous tothe familiar

process employed for instrumentsusing cuvettes. Exact pipetting is of

courseaprerequisiteto ensureuniformfilling heights (Fig. 2B).

The results from the factor-basedmethod are compared withresults

evaluated via standard curve and withcuvette measurements carried out inareference spectrophotometer.

Material

>UltraPure™Herring Sperm DNA Solution(Life Technologies®;

cat. no. 15634-017)

>Quar tzgl asscuv ettewi tha10mmpathlength(Hellma ®Analytics)

>Referencespectrophotometer

>Eppendorf MicroplateUV-VISwithUVtransparent foilbottom

(Eppendorf)

>Eppendorf PlateReaderAF2200

(Eppendorf)

>Tr i sbuffer : 0. 1M, pH8. 0

Method

The following DNAconcentrations are

prepared : 5, 10, 25, 50, 60, 80 and

Lightpath

L i gh t p a t h

L i gh t p a t h

Volume Volume

Cuvette

M icr op la te M ic ro pl at e

d

d

100ng/µL.Everydilutionstepis checked

ina quartzglass cuvette ina spectro-photometerata260 nmwavelength.The

dilutionsteps 5, 25, 50 und 100 ng/µLare used for preparing the standard

curve(PlateReadermethod»UV260 nmwithstandards«). The concentrations

80 ng/μL, 60 ng/μL and 10 ng/μL arethendeterminedviathe standardcurve.Subsequently, all concentrations are

againdetermined using the factor-based method (PlateReader method

»UV260nm withfactor«)andcomparedtotheresults obtainedwiththes tandard

curve, as well as withthose obtainedfrom the cuvette measurements on

the reference spectrophotometer. Allmeasurements(factorandstandard)

are performed with100, 200, 300 µL(one plate for eachvolume).

During the course offactor-based

measurements, absorbance values atthe following wavelengths are deter-

mined: 260 nm, 280 nm and 340 nm.The value generated at 340 nm is taken

intoconsiderationas a reference valueinorder tominimize the influence of

contamination. The value obtained at280 nm is used tocalculate the A260/

A280 ratioand todetermine the purityofthe DNA. Pure DNAshows a ratio

between1.8 and 2.0. Lower ratios mayindicate proteincontamination. The

absorbance measurement at 260 nm isused tocalculate the DNAconcentra-

tionfollowing Lambert-Beer's law.

Calculationofconcentrationis per-formed bythe instrument's software

byusing the sample specificextinctionfactor of50 for double-stranded DNA

(dsDNA).

A B

d

A= ε· c· d

Fig. 1: Light beam throughthe cuvette

Fig. 2: Photometricdetermination ina microplate. The pathlengthis determined bythe filling height.A) Different liquid levels due tounreproducible pipetting or highvariances in thewell geometryB) Photometricdeterminationinplate readersrequiresidenticalliquidlevels.

A= Absorbance, ε = Extinctioncoefficient,c= concentration, d = optical pathlength

Eppendorf Tubes® 5.0 mL:The »Missing Link«> Eppendorf Reference® 2: The New Legend

> New BioBLU® 1-Liter Single-Use Bioreactors

> Ergonomics in Today's Laboratory Routine

Application Notes

Comparative Run Time Evaluations of PCR Thermal Cyclers · Quantification of Nucleic AcidsUsing a Factor-Based Method in the Eppendorf PlateReader AF2200 · etc.

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Imprint

Editorial teamBerrit Hoff (Editor-in-Chief), Axel Jahns,

Jochen Müller-Ibeler, Natascha Weiß

Publisher

Eppendorf AG, Barkhausenweg 1,

22339 Hamburg, Germany

Telephone: (+49) 40-53801-636

Fax: (+49) 40-53801-840

E-mail: [email protected]

Internet: www.eppendorf.com

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 2014.

Carbon neutrally printed in Germany.

2

Dear Readers,

50 years ago Eppendorf created the Eppendorf Tube and triggered a microtubeevolution. Eppendorf Tubes® are now used every day throughout the world and are arecognized 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, upuntil now there has been no convincing solution for medium sample volumes. Here'sthe 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 to5.0 mL. As you may expect from Eppendorf, we also offer a comprehensive range ofmatching accessories for sample preparation and sample storage – with numerousapplicational benefits. Read more on pages 4 – 5!

Do you already know about the new Multipette® M4*? Its light weight and ergonomicdesign, which was optimized according to the most recent findings, as well as itsintuitive operation enable simple, stress-free and ergonomic dispensing (page 6).

Do you often have to pipette precious liquids? Are extraordinary precision andaccuracy, 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 foryour demanding work. This classic Eppendorf pipette with single-button operation isnow also available as a multi-channel version (page 7).

In addition to further articles on new products, this BioNews edition also includesdetailed Application Notes and a new competition with great prizes.

Enjoy reading!

Your Eppendorf BioNews Editorial Team

EDITORIAL · DEAR READERS

*US/CAN: Repeater® M4

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4 117

(BN 40) JANUARY 2014 PAGE3

Yourlocaldistributor: www.eppendorf.com/contact EppendorfAG · 22331 Hamburg · Germany· E-mail: [email protected] · www.eppendorf.com

Quantification of Nucleic Acids Using a Factor-Based Method

in the Eppendorf PlateReader AF2200TANJAMUSIOL1,BETTINASCHEUMER2,MARKUSLAPCZYNA 2

1EPPENDORFAG,HAMBURG,GERMANY

2EPPENDORFINSTRUMENTEGMBH,HAMBURG,GERMANY

Introduction

Determinationofconcentrationvia asample-specificextinctionfactor is a

commonapproachfor the cuvetteformat, but not for the plate format.

TheunderlyingcalculationisLambert-

Beer's law:

For this calculation, knowledge ofthesample specificfactor, as well as the

exact pathlengthare required. Inthecase ofcuvettes, the pathlengthis

determinedbythe shapeofthe cuvette,as the light beam traverses the cuvette

horizontally(Fig. 1).

Inthecase ofabsorbancemeasurements

ina plate, the light beam traverses thesample vertically(Fig. 2A).

The pathlengthis int his case deter-mined bythe filling height. Since the

filling height is not onlydependent onthe volume but alsoonthe geometry

ofthe well, exact determinationofthefilling height is critical.

Inthis article it will be shown how,

usingtheEppendorfMicroplateUV-VISwithadefinedvolume,the concentration

ofa dsDNAsolutionis determined inthe EppendorfPlateReader AF2200 by

means ofa factor-based method. Sincethe exact dimensions ofthe plate and

its wells are known, the softwarealreadycontains the respective path

lengthsforpre-definedfillingvolumes.Calculationofsampleconcentrationis

performed analogous tothe familiarprocess employed for instruments

using cuvettes. Exact pipetting is of courseaprerequisiteto ensureuniform

filling heights (Fig. 2B).

The results from the fact or-based

method are compared withresultsevaluated via standard curve and with

cuvette measurements carried out inareference spectrophotometer.

Material

>UltraPure ™Herring Sperm DNA Solution(Life Technologies®;

cat. no. 15634-017)

>Quartzglasscuvettewitha10mm

pathlength(Hellma®Analytics)

>Referencespectrophotometer

>EppendorfMicroplateUV-VIS

withUV transparentfoilbottom(Eppendorf)

>EppendorfPlateReader AF2200

(Eppendorf)

>Trisbuffer:0.1M,pH8.0

Method

The following DNAconcentrations areprepared : 5, 10, 25, 50, 60, 80 and

Lightpath

L i gh t p a t h

L i gh t p a t h

Volume Volume

Cuvette

Microplate Microplate

d

d

100ng/µL.Everydilutionstepischecked

ina quartz glass cuvette ina spectro-photometerata260 nmwavelength.The

dilutionsteps 5, 25, 50 und 100 ng/µLare used for preparing the standard

curve(PlateReadermethod»UV 260nmwithstandards«). The concentrations

80 ng/μL, 60 ng/μL and 10 ng/μL arethendeterminedviat hestandardcurve.

Subsequently, all concentrations areagaindetermined using the factor-

based method (PlateReader method»UV260nmwith factor«)andcompared

tothe resultsobtainedwiththe standardcurve, as well as withthose obtained

from the cuvette measurements onthe reference spectrophotometer. All

measurements(factorandstandard)are performed with100, 200, 300 µL

(one plate for eachvolume).

During the course offactor-based

measurements, absorbance values atthe following wavelengths are deter-

mined: 260 nm, 280 nm and 340 nm.The value generated at 340 nm is taken

intoconsiderationas a reference valueinorder to minimize the influence of

contamination. The value obtained at280 nm is used tocalculate the A260/

A280 ratioand todetermine the purityofthe DNA. Pure DNAshows a ratio

between1.8 and 2.0. Lower ratios mayindicate proteincontamination. The

absorbance measurement at 260 nm isused tocalculate the DNAconcentra-

tionfollowing Lambert-Beer's law.

Calculationofconcentrationis per-

formed bythe instrument's softwarebyusing the sample specificextinction

factor of50 for double-stranded DNA(dsDNA).

A B

d

A = ε· c· d

Fig. 1: Light beam throughthe cuvette

Fig. 2: Photometricdeterminationina microplate. The pathlength is determined bythe filling height. A) Different liquid levels due tounreproducible pipetting or highvariances inthewell geometryB) Photometricdeterminationinplatereadersrequiresidenticalliquidlevels.

A= Absorbance, ε = Extinctioncoefficient,c= concentration, d = optical pathlength

3CONTENTS

KHANDAKER SIDDIQUEE, MA SHA

A Novel Method for the Expansion of Mesenchymal Stem CellsUsing the Eppendorf New Brunswick™ S41i CO2 Incubator Shaker

1 – 2

TANJA MUSIOL, BETTINA SCHEUMER, MARKUS LAPCZ YNA

Quantification of Nucleic Acids Using a Factor-Based Methodin the Eppendorf PlateReader AF2200

3 – 4

NATASCHA WEISS, FRAUKE GOTZHEIN

Comparison of Eppendorf Tubes® 5.0 mL to Conical 15 mL Tubesin Regard to Releasable UV-Absorbing Substances (Leachables)

5 – 6

NILS GERKE

Comparative Run Time Evaluations of PCR Thermal Cyclers 7 – 8

IN THE SPOTLIGHT Eppendorf Tubes® 5.0 mL: The »Missing Link« 4 – 5

INNOVATION Your Sample is Safe with ep Dualfilter T.I.P.S.® SealMax 8

»Smooth Operators«: New Micromanipulators 11

STRAIGHT FROM THE LAB 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

SERVICE Prize Competition 14

Reply Fax / Readers' Service 15

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4

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«.

IN THE SPOTLIGHT · EPPENDORF TUBES® 5.0 ML: THE »MISSING LINK«

Eppendorf Tubes® 5.0 mL:

The »Missing Link«

BERRIT HOFF, EPPENDORF AG

In concert with a unique system of match-

ing accessories for sample preparation andstorage, a comprehensive and thought-outsolution is now available. More detailsdirectly from Dr. Daniel Wehrhahn (GlobalProduct 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, thesevessels often lack important featuressuch as high centrifugation stability or asafe seal. This is why users mainly

resorted to the tried and tested 15 mLconical tubes.

What sets the new tube apart?

For one, comfortable handling: thanks toits snap cap lid, our tube can be effort-lessly opened and closed with one hand.The handling of screw cap tubes is lesscomfortable. Second, better protectionagainst cross-contamination. Especiallyduring sample processing of small vol-umes in large, conical tubes, the pipettecone is often inserted deeply into thetube 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

pipettes, are used, which is less ergo-nomic, especially when working in asterile hood. Last, but not least, ourextensive selection of accessories, whichallow the use of the tube in almost anycontext.

Which material is used for the production of

the 5.0 mL Tube?

An especially clear and high quality poly-propylene. It offers high transparency forgood sample visibility while simultane-ously minimizing leaching. Therefore,the user does not have to worry aboutsubstances leaching from the plasticwhich may influence the experiment.In comparison with 15 mL conical tubes,the Eppendorf Tubes 5.0 mL do verywell in this category (editor's note: pleasealso refer to the Application Note onpages 5 – 6).

Furthermore, the tubes feature high

chemical resistance as well as tempera-ture stability and mechanical stability.

How stable are the tubes?

Eppendorf Tubes 5.0 mL were designedfor safe and stable centrifugation up to25,000 x g, thus preventing sample losswhen performing quick protocols. Thiscentrifugation stability also exceeds thatof 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

The Eppendorf Tube 5.0 mL closes the ga p between microcentrifuge tube and conical screw ca p vessel.

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Tip

Eppendorf Tubes®

5.0 mL – systemati-

cally simple and safe

> Simple, practical and ergonomic single-

hand operation > Hinged lid for minimized sample evapo-

ration 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 accessoriesavailable: 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 atwww.eppendorf.com/5mL

5EPPENDORF TUBES® 5.0 ML: THE »MISSING LINK« · IN THE SPOTLIGHT

Clip 5.0 mL« ensures that the tubesremain securely sealed.

Which purity grades are available for the

Eppendorf Tube 5.0 mL?

In addition to the established puritiesEppendorf Quality™, PCR clean andEppendorf Biopur® we also offer thepurity grade »Sterile«. These productsare additionally tested for the absence ofpyrogens. Owing to the variety of certifiedpurity grades, the 5.0 mL tubes aresuited for all types of different methodswithin cell biology and molecular biologylaboratories. Furthermore, the specialEppendorf LoBind® material, offered inthis format for the first time, makes them

ideal for work with sensitive nucleic acidor protein samples.

It is noticeable that the 5.0 mL Tube also

features a conical shape.

Integrating the tube seamlessly into theexisting workflow, and ensuring its com-patibility with instruments and accessoriesdesigned for 15 mL conical tubes whichare already present in the laboratory wasimportant to us. This way, the user cancontinue to use many adapters and racksin a cost-saving fashion.

At the same time, we have optimized ourown selection of instruments and acces-

Optimized adapters for existing rotorsPerfect mixing, heating and cooling with the Thermomixer® C

Precise one-step pipetting with Eppendorf pipettes andepT.I.P.S.® 5.0 mL

sories: we offer matching high-speedrotors for our Centrifuges 5430/R, 5427 R,5804/R and 5810/R.

In addition, adapters and inserts for allexisting Eppendorf rotors with bores for15 mL or 50 mL vessels are available. Fortemperature control and mixing, 5.0 mLinterchangeable blocks for the Eppen-dorf ThermoMixer® C and Thermomixercomfort, the ThermoStat C and theThermoStat plus, are available. Even a5.0 mL solution for the automatic pipet-ting system epMotion® was included.

For which methods are the Eppendorf

Tubes 5.0 mL especially recommended?

Larger volumes of culture media areadvantageous for a multitude of applica-tions. For example, when isolating certaintypes of plasmid DNA considerably higheryields may be achieved in fewer stepswhen the volume of the starter culture isincreased 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 noticevery high demand by our users. Our»missing link«, and the holistic approachof our system, has obviously struck bull'seye. Considering all this, both projectsare comparable. This validates and alsopleases us!

[1] APPLICATION NOTE No. 262/ June 2013(www.eppendorf.com/application)

Eppendorf Tubes® 5.0 mL • Ref. no. 264

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6 NEWS · DISPENSING NEWLY DEFINED: SIMPLE, STRESS-FREE, ERGONOMIC

Dispensing Newly Defined:

Simple, Stress-Free, Ergonomic

SABINE KÜHN, EPPENDORF AG

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.

Reliable system for sophisticatedchallenges

In combination with the dispensing tipsCombitips advanced, the Multipette M4functions according to the direct displace-ment principle. The liquid is dispensedwithout an air cushion, in direct contactwith the Combitip's piston. Whether youwork with viscous solutions (e.g. glycerol),liquids with high vapor pressure (e.g.ethanol) or other problematic substances:independent of density, viscosity orvolatility of the liquid, the correct volume

is dispensed every time. Accurately,swiftly and free from contamination.

The new Multipette M4 facilitates and accelerates the perfor-mance of long pipetting series.

Swift and stress-free work

The Multipette M4 makes a considerablecontribution to the simplification as wellas more rapid completion of long pipettingseries, be it for aliquoting, when usingkits, or for filling reaction tubes and plates.This way, one filling of the Combitip maybe dispensed in up to 100 individual steps– a comfortable solution for filling 96-wellplates, to name just one application.

The new integrated step counter dis-plays the dispensing steps completed.This feature ensures error-free dispens-ing in the face of work interruptions ordistractions. Speaking of work interrup-tion: The new sleep function turns theMultipette M4 off when not in use, thusensuring energy consumption that iseasy on the battery!

Combitips advanced: the perfect

system component

The Multipette M4 offers a broadvolume spectrum, dispensing in stepsfrom 1 μL up to 10 mL.

To this end, Combitips advanced areavailable in 9 sizes from 0.1 mL to 50 mL.Even the variety of volumes leaves nowish unfulfilled: each Combitip allows 20different dispensing volumes. Followinginsertion of the Combitip, the Combitip-size is recognized by a sensor. The dis-pensing volume selected via the volumeselection wheel appears automatically onthe display.

After use, the Combitip advanced issimply ejected and disposed of by one-handed button operation, without anycontact or danger of contamination.

Learn more

All details are available atwww.eppendorf.com/m4 or in the current brochure(simply order by reference number).

Everything under control:dispensing volume andnumber of steps

The right tip for each application: Combitips advanced areavailable in 9 volume sizes and 3 purity grades.

Multipette® M4 • Ref. no. 268

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7EPPENDORF REFERENCE® 2: THE NEW LEGEND · NEWS

Eppendorf Reference® 2:

The New Legend

CHRISTIANE MARKAU, EPPENDORF AG

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.

Single-button operation is the hallmarkof the Reference 2. Volume selection,liquid aspiration and dispensing, as wellas tip disposal, occur with just one

button – fast and ergonomic.The pipette achieves quick tip ejectionwith active aerosol reduction. No lateralmovement of the thumb is required fortip ejection, thus reducing the risk ofrepetitive strain injuries (RSIs).

NEW! Improved traceability

Security is provided by the integratedRFID chip, on which important pipettedata such as serial number and factorycalibration dates are irrevocably saved.Using the Eppendorf TrackIT RFID reader,these data can be read and also supple-mented with additional information suchas inventory number, calibration due dateand location. The Eppendorf TrackITsoftware supplies an overview of all dataentered and optionally saves each changein a defined location where it may be readinto a LIMS system. In addition, the serialnumber of the Reference 2 is printed onboth the upper and the lower parts ofthe pipette. This way, replacement orexchange of the lower part becomesimmediately obvious.

Reproducibly high quality results

The spring-loaded tip cone ensures low

operating and ejection forces, as well asreproducible, user-independent tip fit.For the new multi-channel variants, thenew channel indicator guarantees acontinuously identical pipette alignmentthroughout the work process.

A premium pipette for highest

expectations

Only state of the art technologies andselect materials have been introducedinto the Reference 2. It is completelyautoclavable, it offers high reproducibilityand reliability. It is therefore the idealinstrument for working with valuableliquids, as well as for all applicationsrequiring maximum accuracy.

Endurance test for the new legend

The new Reference 2 is as long lived androbust as its predecessor. The proof isdelivered by our short film »The NewLegend: The outstanding robustness ofthe Eppendorf Reference® 2 pipette«.

It shows the Reference 2 pass a veryspecial endurance test. The film andfurther information are available atwww.eppendorf.com/reference2.

We would be pleased to send you thedetailed brochure; just order by refer-ence number.

Eppendorf Reference® 2 • Ref. no. 269

Eppendorf Reference 2: the new reference class for modernliquid handling tools

The name »Reference« stands for extraordinary precision and accuracy, a long service life, and an ergonomic design.

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8 INNOVATION · YOUR SAMPLE IS SAFE WITH EP DUALFILTER T.I.P.S.® SEALMAX

Your Sample is Safe with

ep Dualfilter T.I.P.S.® SealMax

BRIGITTE KLOSE, EPPENDORF AG

Therefore, some filter tips also provideprotection against liquids. The filters forthese tips contain sealing or barrier-forming additives. As soon as they comeinto contact with the sample liquid, thefilters swell and prevent the liquid frompassing through the filter.

The ability to recover as much sample aspossible thereafter is questionable andoften it cannot be excluded that the filter

material, after having been in contact withthe sample, may negatively affect theefficiency of classic PCR and quantitativereal-time PCR.

»SealMax« stands for maximum pro-

tection against contamination and

sample loss

The new violet/white filter of the epDualfilter T.I.P.S. SealMax filter tipscombines reliable pipette protectionagainst contaminating contact with thesample due to accidental over-pipetting

with virtually 100 % protection againstaerosols and biomolecules.

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 configu-

rations 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.

10 µL Filter t ip 200 µL Filter t ip

100%

90%

80%

70%

60 %

50%

40%

30%

20%

10%

0%

S a m p l e r e c o v e r y

ep Dualfilter T.I.P.S.SealMax

Competitor G Competitor A

This innovative filter is the first Dualfilterwhich immediately closes when it comesinto contact with the sample, withoutinhibiting it. Sample loss is thus avoidedwhen working with aqueous solutionshaving physical properties like water.In these cases the pipetting procedurecan continue unimpeded without wastingtime. Virtually all of the aspirated aqueous

sample material can be recovered.Two layers for effective measurement

The violet/white filter which is made offlexible material also has different poresizes in its two layers for retainingaerosols and biomolecules.

Like the established ep Dualfilter T.I.P.S.with a blue/white filter, the air flow rate isnot affected. The filter penetration ratewas measured according to EN 1822with 99.5 % retention of aerosols, which

complies with HEPA (High EfficiencyParticulate Airfilter) class E12. A studycarried out by Eppendorf AG determinedthe sample recovery rate of ep DualfilterT.I.P.S. SealMax and other self-sealingfilter tips following over-pipetting ofwater. The recovery rate was significantlyhigher than those of the competitorproducts tested (Fig. 1).

More information

ep Dualfilter T.I.P.S. SealMax are avail-able in the tested purity grade PCRclean/Sterile (sterile and pyrogen-free)in volume ranges from 10 µL to 1,000 µL.Batch-specific certification is conductedby an independent laboratory.

You can request the new brochurequoting the reference number or youcan inform yourself online atwww.eppendorf.com/consumables .

ep Dualfilter T.I.P.S.® SealMax • Ref.no. 262

Fig. 1: A comparison of the samplewater recovery rate of ep Dualfil terT.I.P.S. SealMax with the self-sealingfilter tips of two competitors.The figure shows the average samplerecovery rates in % collected from10 individual measurements.

Error bar = standard deviation.

Source: Application Note 273.*Determination of the sample recoveryrate of self-sealing filter tips in case ofover-pipetting of water.

*Available for download as a PDF atwww.eppendorf.com/applications

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(BN 40) JANUARY 2014 PAGE 1

Your local distr ibutor: w ww.eppendorf.com/contac t Eppendorf AG · 22331 Hamburg · Germany · E-mail: eppendorf@ eppendorf.com · www.eppendorf.com

A Novel Method for the Expansion of Mesenchymal Stem Cells

Using the Eppendorf New Brunswick™ S41i CO2 Incubator Shaker

KHANDAKER SIDDIQUEE & MA SHA, EPPENDORF, INC., ENFIELD, CT, USA

Introduction

The expansion of stem cells, includingmesenchymal stem cells (MSCs), hasbeen successfully demonstrated usingmicrocarrier-based small bioreactorssuch as spinner flasks. In this study, weexplored a simple alternative to theMSC expansion in spinner flasks usingconventional shake flasks in a CO2 incu-bator with built-in shaking capability:the Eppendorf New Brunswick S41i.

Here we compared microcarrier-based12 days expansion of adipose-derivedmesenchymal stem cells (AdMSCs) inspinner flasks to shake flasks analyzingparameters like cell growth, metabo-lism as well as quality and differentia-tion abilities of the cells.

Cells on microcarriers were counted by

a hemocytometer. The supernatantscollected during cell counting were usedfor further biochemistry and metabolitemeasurements by an automated bio-chemistry analyzer. To assess the qualityof AdMSCs after expansion and toconfirm that the stem cell markers wereretained during the microcarrier-basedculture, CD44 and CD90-specific fluo-rescent immunoassays were performed.

To prove their ability to differentiate,adipocyte and osteocyte differentiationassays were performed. Adipocyte and

osteocyte differentiated cells wereidentified by cell-type specific stainingwith either Oil red O or Alizarin red S.

For more detailed information on proce-dure and instruments used please seeApplication Note 259 (document availableonline at www.eppendorf.com/

applications.)

Results and discussion

Cell growth studies revealed thatAdMSCs cultured under shake flaskconditions achieved excellent growthduring the 12 day batch culture (Fig. 1A).

Biochemistry and metabolite analysisrevealed that glucose concentrations

decreased from 1.09 g/L to 0.548 g/L

(for shake flask culture) and 0.798 g/L(for spinner culture), whereas lactateconcentrations increased from 0.042 g/Lto 0.396 g/L (for shake flask culture)and 0.259 g/L (for spinner culture)after 12 days of culture (Fig. 1B &C).

The higher glucose consumption andlactate production rate seen in the shakeflask culture supports the finding thatthe stem cells grew at a faster rate underthe shake flask conditions. Furthermore,during early growth phase (day 4); theamount of ammonium accumulated

in spinner flask culture (2.4 mM) was1.8-fold higher than in shake flaskculture (1.3 mM) (Fig. 1D).

It has been shown that even low levelsof ammonium (1.9 mM) inhibit MSCgrowth. The spinner culture has shownammonium levels exceeding 2 mM earlyand throughout the culture process,which indicates the slower growth bythe spinner method could be a result ofammonium toxicity-induced growthinhibition.

The fact that the spinner culture hadelevated ammonium levels early in theculture (not seen in the shake flask) alsoindicates possible stem cell damage

C e l l n u m b e r ( 1 0 4 ) / m L

G l u c o s e c o n c e n t r a t i o n ( g / L )

L a c t a t e c o n c e n t r a t i o n ( g / L )

N H 3 C o n c e n t r a t i o n ( m M )

6

5

4

3

2

1

0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

0.450.400.350.300.250.200.150.100.050.00

1.2

1.0

0.8

0.6

0.4

0.2

0.0

Growth curve of AdMSCs

Concentration of lactate in AdMSC cultures

Concentration of glucose in AdMSC cultures

Concentration of NH3 in AdMSC cultures

Day

DayDay

Day0

00

01

11

12

22

23

33

34

44

45

55

56

66

67

77

78

88

89

99

910

1010

1011

1111

1112

1212

12

A B

C D

Materials and methods

AdMSCs were initially seeded in bothsystems at a density of 3 x 103 cells/cm2 in appropriate medium. For the initial

attachment of cells on the microcarriers(0.5 g of 125 – 212 micron) in both sys-tems, the agitation speed of the NewBrunswick S41i CO2 incubator shakerand rotation speed of the spinner wereboth kept at 50 rpm and incubated for2 h at 37 °C with 5 % CO2.

After that the volume was adjusted to50 mL total volume and the speed inboth systems was raised to 70 rpm.Cell culture studies were conducted for12 days and samples were collected forcell growth, biochemistry and metabo-

lite analysis daily.

The New Brunswick S41i CO2 Incubator Shaker, designedfor both non-adherent and adherent cell culture applications,combines the precise temperature and CO2 control of anincubator with the reliable New Brunswick laboratory shakerdrive mechanism. Key features include sealed inner/outerdoors, high-temperature disinfection, and reduced CO2 consumption compared to competitor models [1].

Fig.1: Analysis of AdMSCs growth and metabolism in shake flask and spinner flasks culture conditionsA) Growth B) Glucose utilization C) Lactate production D) Ammonium productionBlue: Shake flask Red: Spinner flask

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PAGE 2 (BN 40) JANUARY 2014


A Novel Method for the Expansion of Mesenchymal Stem Cells

Using the Eppendorf New Brunswick™ S41i CO2 Incubator Shaker

due to shear force by the spinner rod.Immunostaining of stem cell surfacemarkers revealed that AdMSCs retained

stem cell surface markers during growthunder shake flask culture condition(Fig. 2A&B).

Adipocyte and osteocyte differentiationassays indicated that most of the AdMSCsfrom shake flask culture differentiatedinto either adipocytes or osteocytessuccessfully (Fig. 3 A&B).

A

A

B

B

Conclusion

Stem cell expansion using shake flaskconditions appears to be a viable andsimple alternative to the spinner flasksystem.

The New Brunswick S41i reducesshearing, eliminates potential celldamage by the spinner rod, decrease

the risk of contamination associatedwith inserting a magnetic stirrer baseinto the CO2 incubator and reducesexperimental complexity.

This method also greatly increases thecell culture capacity whereby a largenumber of shake flasks can be placed inthe New Brunswick S41i simultaneously.In the case of spinner flask culture, atypical incubator without active coolingcan only handle the heat emitted froma very limited number of magneticstirrer bases before causing tempera-

ture setpoint overshoot, a significant

limitation to the scale-up potential ofthe spinner method. This reinforces thesuperiority of the New Brunswick S41iCO2 Incubator Shaker as an alternativeto incubator/spinner based stem cellculture.

This method eliminates a scale-upbottleneck while providing highestquality stem cell culture for inoculation

of large scale industrial bioreactors forthe production of clinical material.The shake flask has lower cost and lessparts to disassemble, clean, assembleand autoclave.

Literature

[1] Kohlstrom et al. (2012): Hybridoma andCHO Cell Culture using the New Brunswick™ S41i, an Environmentally-Friendly, Low EmissionIncubator Shaker. Application Note 255.

Available online at www.eppendorf.com/applications

CD90 marker (red)

Nucleus (blue)

Microcarrier bead

CD44 marker (green)

Nucleus (blue)

Microcarrier bead

200 µm200 µm

Fig. 2: Stem cell marker identification assay for AdMSCs expanded on microcarriers in shake flaskA) 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 A lizarin Red S positive staining.

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• Ref. no. 253

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in the Eppendorf PlateReader AF2200

TANJA MUSIOL1, BETTINA SCHEUMER2, MARKUS LAPCZYNA2 1EPPENDORF AG, HAMBURG, GERMANY

2EPPENDORF INSTRUMENTE GMBH, HAMBURG, GERMANY

Introduction

Determination of concentration via asample-specific extinction factor is acommon approach for the cuvetteformat, but not for the plate format.

The underlying calculation is Lambert-Beer's law:

For this calculation, knowledge of thesample specific factor, as well as theexact path length are required. In thecase of cuvettes, the path length isdetermined by the shape of the cuvette,as the light beam traverses the cuvettehorizontally (Fig. 1).

In the case of absorbance measurementsin a plate, the light beam traverses thesample vertically (Fig. 2A).

The path length is in this case deter-mined by the filling height. Since thefilling height is not only dependent on

the volume but also on the geometryof the well, exact determination of thefilling height is critical.

In this article it will be shown how,using the Eppendorf Microplate UV-VISwith a defined volume, the concentrationof a dsDNA solution is determined inthe Eppendorf PlateReader AF2200 bymeans of a factor-based method. Sincethe exact dimensions of the plate andits wells are known, the softwarealready contains the respective pathlengths for pre-defined filling volumes.Calculation of sample concentration isperformed analogous to the familiarprocess employed for instruments

using cuvettes. Exact pipetting is ofcourse a prerequisite to ensure uniformfilling heights (Fig. 2B).

The results from the factor-basedmethod are compared with resultsevaluated via standard curve and withcuvette measurements carried out in areference spectrophotometer.

Material

> UltraPure™ Herring Sperm DNASolution (Life Technologies®;cat. no. 15634-017)

> Quartz glass cuvette with a 10 mmpath length (Hellma® Analytics)

> Reference spectrophotometer

> Eppendorf Microplate UV-VISwith UV transparent foil bottom(Eppendorf)

> Eppendorf PlateReader AF2200(Eppendorf)

> Tris buffer: 0.1 M, pH 8.0

Method

The following DNA concentrations areprepared : 5, 10, 25, 50, 60, 80 and

Light path

L i gh t p a t h

L i gh t p a t h

Volume Volume

Cuvette

Microplate Microplate

d

d

100 ng/µL. Every dilution step is checkedin a quartz glass cuvette in a spectro-photometer at a 260 nm wavelength. Thedilution steps 5, 25, 50 und 100 ng/µLare used for preparing the standardcurve (PlateReader method »UV 260 nmwith standards«). The concentrations80 ng/μL, 60 ng/μL and 10 ng/μL arethen determined via the standard curve.Subsequently, all concentrations areagain determined using the factor-based method (PlateReader method»UV 260 nm with factor«) and compared

to the results obtained with the standardcurve, as well as with those obtainedfrom the cuvette measurements onthe reference spectrophotometer. Allmeasurements (factor and standard)are performed with 100, 200, 300 µL(one plate for each volume).

During the course of factor-basedmeasurements, absorbance values atthe following wavelengths are deter-mined: 260 nm, 280 nm and 340 nm.The value generated at 340 nm is takeninto consideration as a reference valuein order to minimize the influence ofcontamination. The value obtained at280 nm is used to calculate the A260/ A280 ratio and to determine the purityof the DNA. Pure DNA shows a ratiobetween 1.8 and 2.0. Lower ratios mayindicate protein contamination. Theabsorbance measurement at 260 nm isused to calculate the DNA concentra-tion following Lambert-Beer's law.

Calculation of concentration is per-formed by the instrument's software

by using the sample specific extinctionfactor of 50 for double-stranded DNA(dsDNA).

A B

d

A = ε · c · d

Fig. 1: Light beam through the cuvette

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 th ewell geometry B) Photometric determination in plate readers requires identical liquid levels.

A = Absorbance, ε = Extinction coefficient,c = concentration, d = optical path length

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This factor states that an absorbancevalue of 1, measured in a 10 mm lightpath, translates to a DNA concentrationof 50 μg/μL [1].

Results

Figures 4, 5 and 6 show the respectivestandard curve obtained by 100, 200and 300 µL measurement. All standardcurves show optimal linearity. Table 1gives an overview of the factor-basedand standard curve based measure-ments. In addition the correspondingresults from the cuvette measurementswith the reference spectrophotometerare displayed. Same or even better arethe results obtained with the factor-based measurements compared tomeasurements with standard in relationto the reference measurements per-formed in cuvettes.

Conclusion

As clearly shown by the comparativeanalyses of measurement results, factor-based determination of sample concen-trations is possible in the EppendorfPlateReader AF2200 when using theEppendorf Microplate UV-VIS. Theresults are comparable amongst each

other, as well as against referencemeasurements in standard cuvettes.The same is true for the comparisonof factor-based results with analysesvia standard curves, which representthe common method for concentrationdetermination in a plate reader. Sincefilling height determines the path length,which is a fundamental parameter ofconcentration determination, highlyreproducible dispensing of samplevolumes is critical, as well as the use ofplates with known and consistent well

geometry.

Literature

[1] Gallagher, Sean R. (2001), Quantification ofDNA and RNA with Absorption and FluorescenceSpectroscopy, Current Protocols in Cell Biology,Appendix 3D

Splashes on the rim of the well or theformation of a pronounced meniscus(e. g. resulting from detergent contain-ing buffers) are to be avoided. Takentogether, it is safe to state that factor-based calculation of the concentrationin the Eppendorf PlateReader AF2200,in combination with the EppendorfMicroplate UV-VIS, represents aqualitative substitute to the commonmethod of extrapolation from a standard

curve. By eliminating the effort re-

quired to generate a standard curve, thismethod is very economical compared tothe current common procedures, sinceboth time and money are saved.

A b s o r b a n

c e

0.70000.60000.5000

0.40000.30000.20000.10000.0000

y = 0.0067x - 0.0015 R²= 1.0000

Concentration [µg/mL]

0.000 20.000 40.000 60.000 80.000 100.000 120.000

A b s o r b a n c e

1.5000

1.0000

0.5000

0.0000

y = 0.0137x - 0.0066 R²= 1.0000


0.000 20.000 40.000 60.000 80.000 100.000 120.000

A b s o r b a n c e

2.5000

2.0000

1.5000

1.0000

0.5000

0.0000

y = 0.0202x - 0.0085 R²= 1.0000


0.000 20.000 40.000 60.000 80.000 100.000 120.000


in the Eppendorf PlateReader AF2200

PrepareddsDNA con-centration in

[µg/mL]

Cuvettesbased mea-surements

performedin a spectro-photometer(references)

[µg/mL]

Filling volumein the plate

[µL]

Results ofstandard

curve based

measurementsperformed in

the MicroplateUV-VIS[µg/mL]

Results offactor-based

measurements

performed inthe Microplate

UV-VIS[µg/mL]

Variancebetween stan-

dard curve

and cuvettesbased mea-surements

[%]

Variancebetween

factor and

cuvettesbased mea-surements

[%]

Variancebetween

factor and

standardcurve based

measurements[%]

10 10.1 100 10.25 9.75 1.5 -3.4 5.1

60 61.9 100 59.5 59.18 -4.0 -4.6 0.5

80 82.7 100 78.96 79.31 -4.7 -4.3 -0.4

10 10.1 200 9.91 9.58 -1.9 -5.4 3.4

60 61.9 200 59.73 60.12 -3.5 -3.0 -0.7

80 82.7 200 79.3 80.72 -4.3 -2.5 -1.8

10 10.1 300 10.25 9.72 1.5 -3.9 5.5

60 61.9 300 59.63 60.75 -3.7 -1.9 -1.9

80 82.7 300 79.21 81.1 -4.2 -2.0 -2.4

Fig. 4: 100 µL measurement; standard curve



Table 1: Summary of factor-based and standard curve based measurements of dsDNA performed in the EppendorfPlateReader AF2200 using the Eppendorf Microplate UV-VIS, compared to measurements performed in cuvettes usinga spectrophotometer

Fig. 3: Screenshot of the method »Nucleic Acid quantifi-cation (UV 260 nm with factor)« from the software of thePlateReader AF2200. (Example: measurement of 100 µL).

Readers' service

Eppendorf PlateReader AF2200 • Ref. no. 261

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Comparison of Eppendorf Tubes® 5.0 mL to Conical 15 mL Tubes

in Regard to Releasable UV-Absorbing Substances (Leachables)

Abstract

Substances which are released fromplastic consumables (»leachables«)may have an influence on laboratoryexperiments. If they absorb light in theUV range, they can easily be detectedby heating water in reaction vessels,followed by photometric analysis. Suchsubstances, for example, can easilyfalsify DNA measurements.

The present Application Note describesexperimental comparison of Eppendorf

Tubes 5.0 mL and conical 15 mL ves-sels according to this method. It canbe shown that a significant amount ofleachables is released from the differ-ent 15 mL vessels. In contrast, forEppendorf Tubes (and a glass controlvessel) the extinction is so low thatphotometric detection methods are notcompromised.

Introduction

The topic of »leachables« (substancesleaching from plastics) has been in the

news for some years, and it is especiallysignificant in the fields of food packagingand beverage containers [1, 2].

In 2008 a publication in Science® broughtto light the problems associated withplastic consumables in laboratories. Inthis and other publications the influenceof different additives which may leachfrom plastic containers and pipette tipson enzyme activity was demonstratedby means of specific enzyme assays[3, 4, 5, 6].

Even standard laboratory methodssuch as photometric detection ofnucleic acids and proteins can befalsified by leachables. Using a simpletest system which includes heating ofwater in reaction vessels (with standardlaboratory methods), followed by anabsorbance scan in the UV range, itcould be demonstrated that UV-absorb-ing substances leach from single useproducts [7].

In this publication, as well as in theEppendorf Application Note 235 [8] it

was further shown that water samples

thus obtained from Eppendorf reactionvessels did not have significant adverseeffects on photometric analyses. Theexperiments in the present ApplicationNote were conducted in accordancewith the test described above [7, 8].

The aim of this study was the compari-son of the Eppendorf Tubes 5.0 mL withconical 15 mL vessels from differentmanufacturers with regards to leachingof UV-absorbing substances.


Three Eppendorf Tubes 5.0 mL and threeconical 15 mL vessels each of four differ-ent manufacturers (B, C, F, V) were filledwith 1 mL water (for molecular biology)and incubated for 30 min at 90 °C* inthe Eppendorf ThermoMixer® comfort,while mixing occurred at 600 rpm.

Similar conditions are found in manyprotocols for sample preparation, suchas sample lysis. Following incubation,samples were allowed to cool down for10 min. The samples were scanned

across the range between 220 nm to340 nm in UVettes, using the Eppen-dorf BioSpectrometer®.

The averages and standard deviationswere determined from the three re-spective replicates. The extinctionsmay be considered proportional to theamount of substances leaching fromthe vessels. Non-incubated water wasused as the blank, while water whichhad been incubated in a glass containerserved as the control. The dsDNA con-centration which could theoretically bederived from the measured values wascalculated from the extinction at 260 nmusing the factor 50 μg/mL per unit of

extinction.Results and discussion

Fig. 1 shows the absorbance spectraof water obtained from the differentvessels in which it had been incubatedfor 30 min at 90 °C while being mixedat 600 rpm. In addition, the insert liststhe dsDNA concentrations theoreticallyderived from the extinctions measuredat 260 nm, which may therefore bemisinterpreted as DNA. A glass controlwas used since no significant leaching

of UV-absorbing material is observedunder the given experimental condi-tions [7].

NATASCHA WEISS1, FRAUKE GOTZHEIN2 1EPPENDORF AG, HAMBURG, GERMANY

2UNIVERSITY OF HAMBURG, ACADEMIC PROGRAM MOLECULAR LIFE SCIENCES, HAMBURG, GERMANY

0.300

0.250

0.200

0.150

0.100

0.050

0.000

Absorbance scan of water following incubation at 90 °C

Wavelength λ [nm]

E x t i n c t i o n [ O D ]

220 240 260 280 300 320 340

Theoretical dsDNAconcentrations

Manufacturer F: 5.01 µg/mL

Manufac turer 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

Manufac turer C Manufac turer B Manufact urer F Manufac turer V

Eppendorf Glass value

Fig. 1: Absorbance spect ra of water following incubation in Eppendor f Tubes 5.0 mL and conical 15 mL vessels by oth ermanufacturers for 30 min at 90 °C

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As expected, the glass vessel producesthe lowest values. The absorbance spec-trum of water obtained from Eppendorfvessels is only slightly higher than thatof the control. The extinction value at260 nm translates to a theoretical dsDNAconcentration of 0.35 μg/mL for the glasscontrol, and to 0.81 μg/mL for the Eppen-dorf Tubes. These values are below therecommended limit of quantification forthe BioSpectrometer (sample extinctionvalues of 0.02 – 0.03 or dsDNA concentra-tion of 1.0 – 1.5 μg/mL, respectively [9]).

Since other influencing factors such asparticles, turbidity or bubbles inside thesolution significantly compromise theresults at very low extinctions, thesedata are considered not meaningful with

respect to the leaching of substances.In contrast, significant absorbancespectra are recognizable in all watersamples which were heated in theconical 15 mL tubes. The absorbancevalues obtained at 260 nm translate totheoretical dsDNA concentrations of2.31 μg/mL to 5.01 μg/mL. It had beenshown previously that standard labora-tory methods during which sampletemperature is elevated (incubation,PCR, centrifugation, sonication) lead toleaching of UV-absorbing substances

from the containers [7, 8].

Comparison of Eppendorf Tubes® 5.0 mL to Conical 15 mL Tubes

in Regard to Releasable UV-Absorbing Substances (Leachables)

This may yield falsely elevated resultsduring photometric analyses of mole-cules such as nucleic acids and pro-teins which are primarily conducted at260 nm – 280 nm. In the case of samplesof low concentration, these errors maycompromise subsequent experiments.Additional problems may be presentedby the variability of these errors.

Fig. 2 shows an excerpt of the rangebetween 230 nm and 280 nm of thesame absorbance scan, with the stan-dard deviations overlaid. The valuesobtained with water from the 15 mLconical vessels show considerablevariation, indicating that the substancescontained in the vessel material leachat different rates. Thus, the error is not

a constant factor but is also subject tolarge variation.

Conclusion

The data presented in this ApplicationNote show that under the influence ofheat, UV-absorbing substances leachfrom the tested conical 15 mL vessels.These leachables can compromise orfalsify downstream analyses such asDNA quantification.

The new Eppendorf Tube 5.0 mL yieldsan extinction which is only slightly

above that of the control and thus

represents a very good alternative fora large volume tube when minimizingthe influence of leachables in sensitive

methods is crucial. Owing to the certifiedomission of problematic additives suchas lubricants, biocides or plasticizers inthe raw material as well as during theproduction process, these vessels arevery well suited for sensitive analyses.This advantage was previously alsodemonstrated for smaller tube formatsprovided 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 mustbe used to prevent tubes from op ening.

Literature

[1] Vandenberg LN, Maffini MV, Sonnenschein C,Rubin BS, Soto AM. Bisphenol-A and the greatdivide: a review of controversies in the field ofendocrine disruption. Endocr Rev 2009; 30:75–95.

[2] Shotyk W, Krachler M, Chen B. Contaminationof Canadian and European bottled waters withantimony from PET containers. J Environ Monit 2006; 8:288–292.

[3] McDonald GR, Hudson AL, Dunn SM, YouH, Baker GB, Whittal RM, Martin JW, Jha A, Ed-mondson DE, Holt A., Bioactive contaminantsleach from disposable laboratory plasticware.

Science 2008; 322(5903):917.

[4] Watson J, Greenough EB, Lett JE, Ford MJ,Drexler DM, Belcastro JV, Herbst JJ, Chat-

terjee M, Banks M. Extraction, identification,and functional characterization of a bioactivesubstance from automated compound-handlingplastic tips. J Biomol Screen 2009; 14(5):566–572.

[5] McDonald GR, Kozuska JL, Holt A. BioactiveLeachates from Lab Plastics. G.I.T. Laboratory

Journal Europe 2009; 13:24–26.

[6] Olivieri A, Degenhardt OS, McDonald GR,Narang D, Paulsen IM, Kozuska JL, Holt A, Onthe disruption of biochemical and biologicalassays by chemicals leaching from disposablelaboratory plasticware. Can J Physiol Pharmacol 2012; 90(6): 697–703

[7] Lewis LK, Robson M, Vecherkina Y, Ji C,Beall G. Interference with spectrophotometricanalysis of nucleic acids and proteins by leachingof chemicals from plastic tubes. BioTechniques 2010; 48(4) 297–302.

[8] Application Note 235: The influence of UV-absorbing substances released from plasticcontainers (leachables) on photometric analyses(www.eppendorf.com/applications)

[9] Operating manual Eppendorf BioSpectro-meter (www.eppendorf.com/manuals)

0.250

0.200

0.150

0.100

0.050

0.000

Absorbance spectra of water with standard deviation

Wavelength λ [nm]

E x t i n c t i o n [ O D ]

230 240 250 260 270 280

Manufact urer C Manufac turer B Manufac turer F Manufact urer V

Eppendorf Glass value

Readers' service

Eppendorf Tubes®

5.0 mL • Ref. no. 264

Fig. 2: Absorbance spectra of water with standard deviations in the range between 2 30 nm and 280 nm

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A

B



Comparative Run Time Evaluations

of PCR Thermal Cyclers

NILS GERKE, EPPENDORF AG, HAMBURG, GERMANY

Abstract

For the purpose of comparing the speedof different thermal cyclers, the isolatedconsideration of heating and coolingramp rates cited in the technical specifi-cations often does not reflect the actualrun times.

An estimate of actual run times basedon these technical ramp rates may thuslead to false conclusions. Whereas theMastercycler® pro S and the Mastercyclernexus GSX1, as expected from theirfast ramp rates, achieved the shortesttotal PCR run times in these evaluations,

some thermal cyclers made by othermanufacturers showed noticeablyslower run times despite similar citedramp rates.

Introduction

Besides control accuracy and tempera-ture homogeneity, the customarytechnical details of a thermal cycler alsoinclude the ramp rate of the thermoblock.The ramp rate, in particular, is not subjectto a uniform standard; instead, manu-facturers 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 theoption of estimating the actualramp rates based on thesediverse statements. Therefore,comparative investigationswere undertaken in order to

evaluate whether the detailspertaining to the ramp ratesstated in the technical specifi-cations are suitable for esti-mating the total run times ofPCR applications on thermalcyclers.


48 positions of a 96-wellplate (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 platewere filled with 25 μL water each.

Fig.3: Screenshot from a Mastercycler pro run protocol, exported as pdf file, from the instrument soft ware (additional information, e.g. user,program details and additional cycler set tings, is not displayed in this section).

Fig. 2: 3-step PCR program for run time determinationA) Start of run time measurement B) End of run timemeasurement

The plate was subsequently sealed withthe Heat Sealing PCR Film (Eppendorf),centrifuged for 1 min at 500 – 1000 × g,placed into the thermal cycler andsubjected to a standard 3-step PCRprogram (Fig. 2).

The run times were determined for theMastercycler pro S, Mastercycler nexusGSX1, Mastercycler nexus gradient andMastercycler nexus, and eight compet-ing thermal cyclers*.

In cases where the respective thermalcycler software allowed for different

temperature control modes or reactionvolume settings, the fastest rampingspeed and/or the lowest volume settingwere chosen. Measurement of total runtime was initiated immediately follow-ing commencement of the first temper-ature step, and it ended immediatelyafter the temperature of the final stephad been reached.

For thermal cyclers which record andsave a detailed run protocol, it waspossible to determine the total runtime following completion of the runby consulting the records.

Apart from the Mastercycler pro andMastercycler nexus models, whose runprotocols may be exported as pdf filesfor documentation purposes subse-quent to the run (Fig. 3), a detailed,exportable record could be obtainedfrom only three of the competingthermal cyclers.

Results and discussion

The evaluation of the ramp rates stated

by the manufacturers, in comparisonwith the empirically determined runtimes, highlighted the fact that isolatedconsideration of ramp rates in accor-dance with technical data is not suit-able for the reliable prediction of theactual run time of a PCR program(Table 1).

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Comparative Run Time Evaluations

of PCR Thermal Cyclers

On the one hand, the thermal cyclersMastercycler pro S (Fig. 4) and theMastercycler nexus GSX1 (Fig. 5)showed, as expected, the shortest totalPCR run times, in accordance with thefast ramp rates cited. On the otherhand, the run times of certain compet-ing thermal cyclers were considerablylonger than would be expected from theramp rates stated in the manufacturers'technical specifications.

It is thus evident that the thermal cy-clers V, T and R were considerably slow-er in their actual PCR run times than theMastercycler nexus gradient and the

Mastercycler nexus, despite the factthat the respective manufacturers hadcited faster ramp rates for these ther-mal cyclers in their technical specifica-tions. It can be assumed that the follow-ing parameters contribute strongly tothe observed discrepancies:

> For the different thermal cyclers themaximum ramp rates stated in thetechnical manuals are reached fordifferent periods of time during theramping process from one tempera-ture to the next – possibly for onlya short time during each rampingphase for certain thermal cyclers.

Table 1: Total run time of a standard 3-step PCR protocol using the fastest settings possible in the instrument sof tware.Due to diverse manufacturers' statements of ramp rates, only the maximum ramp rate which could be found according tothe technical data for an instrument is presented here.

> Temperature control modes or reac-tion volume settings may also exertconsiderable influence on ramping

behavior [1].

This may even lead to the need tore-optimize a reaction following thetransfer of a PCR system from onethermal cycler to another [2].

Conclusion

These comparative investigations haveshown that the isolated considerationof ramp rates often bears limitedmeaningfulness and may even lead tofalse conclusions with regards to theestimation of the actual PCR run timeof a given thermal cycler. For accurateevaluation of the ramping performanceof a thermal cycler it is imperative thatthe manufacturer makes the informa-tion on all relevant parameters, e. g.detailed description of selectabletemperature control modes, availableto the user.

Besides consideration of the technicaldata, for the purpose of an all-encom-passing assessment of the performanceof a thermal cycler it is strongly recom-

mended to test the instrument in ademo-setting with regards to hardware,software and PCR applications.*Tests were performed on one cycler each of every modeltype.

Literature

[1] Application Note 244.www.eppendorf.com/pcr

[2] Hughes S., Moody A. (eds.): PCR.Scion Publishing Limited; 2007.

Fig. 4: Mastercycler pro S with silver block Fig. 5: Mastercycler nexus GSX1 with silver block

Readers' service

Mastercycler®

Family • Ref. no. 265

Thermal cycler Run time(hh:mm:ss)

Ramp rateaccording to technical data (°C/s)

Mastercycler pro S 00:40:12 6

Mastercycler nexus GSX1 00:42:31 5

C 00:46:50 5

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 00:56:38 3.5

A 01:03:13 3

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9BEST CONDITIONS FOR YOUR PCR · NEWS

Best Conditions for Your PCR

KAY KÖRNER, EPPENDORF AG

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.

Silver block: for higher heating and

cooling rates

The new Mastercycler nexus X1 com-bines the modern and intuitive softwareof the Mastercycler nexus with a fast96-well silver block for higher heatingand cooling rates. The Mastercyclernexus X1 is fast and user-friendly. Itrequires little space and little electricity,and it sends you an email when it is

finished. What more could one ask of aPCR cycler?

Flat block: the solution for unusual

problems

Special applications often require specialconsumables. The Mastercycler nexus flat,with its flat block without wells, offers the

ideal base for slides and other unusualformats of consumables. During in situ PCR the results are typically influencedby the characteristic heat conductivity ofthe respective in situ adapter. Thanks tothe flat block, the Mastercycler nexusflat is capable of heating or cooling yourslides directly, thus foregoing the needfor an adapter.

Universal block for maximum flexibility

The Mastercycler nexus with universalblock can accommodate 96-well PCR

plates, 0.2 mL PCR tubes, 0.2 mL PCRstrips, as well as 0.5 mL PCR tubes.

This feature allows a number of differentassays to be run on the same instrument,which is of advantage to larger researchgroups using different formats of con-sumables.

Regardless of which block you will choose,all instruments of the Mastercycler nexusfamily offer numerous advantages:

> Small footprint > Intuitive graphic programming

> Up to two instruments may beconnected to a central instrument

> Optional gradient

> Email notification

> flexlid® concept: automatic heightadjustment of the lid to accommodatea variety of consumables

> 2 year warranty

> Optional self-test function

> Low noise emission

> Low power consumption

Further information

Additional details are available atwww.eppendorf.com/pcr or in the currentbrochure which may be ordered using thereference number denoted below.

Of course we are always happy to offerin-person consultations.

»To raise new questions,

new possibilities, to regard

old problems from a new

angle, requires creative

imagination and marks real

advance in science.«

ALBERT EINSTEINMastercycler® Family • Ref.no. 265

Silver block (heating r ate approx. 5 °C/s)

Mastercycler nexus with silver block: fast, intuitive, reliable

8/12/2019 BN40_IN_150dpi


News

Correct Pipetting

Pipetting is one of the most important

steps towards a correct analysis result.

However, pipetting technique or mainte-

nance 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 tech-nique 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 pipet-

ting is no coincidence but rather a precisecraft. 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 www.eppendorf.com/

epServices.*

We are also pleased to offer you a personal

consultation.

*The Eppendorf Training Center offers are availablein selected countries only and courses may v ary.

0

Ergonomics in Today's

Laboratory Routine

SABINE KÜHN, EPPENDORF AG

STRAIGHT FROM THE LAB · 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 Inter-national Ergonomics Association IEA,the federation of ergonomics and humanfactors societies around the world, aholistic, ergonomic approach encompassesthe consideration of physical, cognitiveand organizational ergonomics.

This is where the Eppendorf PhysioCareConcept® comes in – an inclusive, ergo-nomic product concept which integratesthe three areas mentioned above in alogical and comprehensible fashion. Inorder to design the workplace/laboratoryunder ergonomic considerations, thePhysioCare Concept differentiatesbetween three core areas – also called»spheres«.

Sphere 1: The user

Sphere 1 focuses on the users' needswith respect to ergonomic productdesign as well as product performance

optimized to their individual needs. It isimportant to Eppendorf that all products,in addition to exceptional ergonomicdesign, ensure simple and intuitivehandling, and that product performancethus satisfies the highest demands.

Sphere 2: The laboratory

Sphere 2 regards the system »laboratory«with respect to harmonic integration ofproducts in line with the specific demandsof the workplace. The resulting chal-lenge for Eppendorf is, for example, to

design instruments with as little noiseemission as possible or to place instrument

ventilation so that neighboring workspaces are not impacted. At the sametime, a well thought-out concept forproduct accessories contributes to thefacilitation of optimized work spaceorganization.

Sphere 3: The workflow

Sphere 3 attends to the actual work flow.

The aim is optimization of laboratoryprocesses, with the final goal of improv-ing results for the entire organization.To this end, Eppendorf supports its userswith a comprehensive selection of consult-ing and services, as well as with educationthrough the Eppendorf Training Center.

Further information

Comprehensive information about thePhysioCare Concept as well as tips andideas for optimizing your laboratoryroutine are available at

http://physiocare.eppendorf.com

The Eppendorf PhysioCare Concept integrates ergonomicsholistically into the modern laboratory routine.

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11

»Smooth Operators«:

New Micromanipulators

HEIDE NIESALLA, EPPENDORF AG

»SMOOTH OPERATORS«: NEW MICROMANIPULATORS · INNOVATION

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.

Thoroughly tested by clients …

During the development phase of theTransferMan 4m and 4r we set great storeby our clients' feedback: in particularrobustness and the new product featureswere evaluated critically during intensiveapplication tests, which were conductedwithin routine applications.

… and approved!

The new TransferMan systems combine

intuitive handling with outstandingprecision.

The exceptionally direct transmission ofmovement in all directions makes usersfeel as though they are working with amechanical system. This feature wasespecially verified by clients who testedthe new instruments in direct comparisonwith mechanic-hydraulic competitors'systems.

The new Eppendorf DualSpeed™ joystickexpands the precise and intuitive directmovement control by an additionaldynamic speed mode in order to coverlonger distances or to accelerate sampleprocessing.

Simplified workflow

Eppendorf micromanipulators are used,among other applications, for artificialinsemination, generation of geneticallymodified animals and plants, as well asfor selection and transfer of single cells,micro-particles and crystals. Thanks to

pre-defined, application-specific userprofiles such as »cell transfer« and »DNAinjection«, the multifunctional instru-ments simplify individual work processes.Intelligent additional functions such assaving of positions are easily accessedwithin the individual profiles.

Flexible application

Thanks to the flexible assembly andinstallation concept, installation of the newmanipulators on all common microscopesis possible. Semi-automatic injections

are enabled by combination with theelectronic Eppendorf microinjectors.

The newly introduced electronic couplingwith the Eppendorf PiezoXpert® enablessemi-automatic piezo-assisted cell pene-tration. This is of interest, for example,for the successful injection of plant cellswhere, on account of the thickness ofthe cell wall, larger distances need to bebridged.

Further information

Detailed Application Notes and additionalinformation about the topic are available atwww.eppendorf.com/cellmanipulation.

Tip: Watch the »new ones« in action onthe Eppendorf YouTube® channel!

TransferMan® 4m/TransferMan ® 4r • Ref.no. 266

The new TransferMan systems combine intuitive handlingwith outstanding precision.

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2 NEWS · NEW 1 LITER VESSELS EXPAND THE BIOBLU® FAMILY

New 1 Liter Vessels Expand

the BioBLU® Family

CLAUDIA HUETHER-FRANKEN, EPPENDORF AG, BIOPROCESS CENTER EUROPE, JUELICH, GERMANY

For users in cell culture who would liketo combine the advantages of single-usetechnology with the proven qualities ofstirred-tank bioreactor design, a uniquelycomprehensive portfolio of stirred rigid-wall single-use bioreactors with workingvolumes from 100 mL to 40 L is nowavailable.

Efficient through parallel processing

The BioBLU 1c/f single-use bioreactorswere developed specifically for use withDASGIP® Parallel Bioreactor Systems

which can operate 4, 8 and more bioreac-tors simultaneously. Users benefit fromparallelizing their processes for cultivationof animal and human cells as well as formicrobial applications with bacteria andyeast.

Reliable and comparable results basedon precise process control, and timesavings by combining parallel workflows,paired with the minimal setup timesrequired for single-use bioreactorssustainably accelerate the development

of bioprocessing. Effectiveprocess planning and auto-mation, design of experiment(DOE) and comprehensivebioprocess informationmanagement additionallymaximize time and costeffectiveness of the process.

Established design combined

with single-use technology

The fully instrumented BioBLU1c/f rigid-wall single-use bio-reactors can be operated withworking volumes between320 mL and 1.25 L for the cellculture variant, and 250 mLto 1.25 L for microbial appli-cations. All critical processparameters such as tempera-ture, pH and soluble oxygencan be monitored and con-trolled with industry standard

sensors. Integrated dip tubesallow controlled addition of

liquids and sampling as well as submersedor headspace-gassing.

The BioBLU 1c comes with two 3 x 45°pitched blade impellers, the BioBLU 1fwith three 6-blade Rushton-type impellers.The specially developed, encapsulatedand magnet-coupled stirrer drive enablessecure and sterile agitation with up to600 rpm in cell culture and 1,600 rpm formicrobial applications.

Mass transfer rates that meet the highdemands of microbial applications areguaranteed. The innovative liquid-freePeltier condenser ensures optimal cooldown of exhaust. In addition, the BioBLU1f is equipped with four innovative cool-ing baffles which support precise tem-perature control even under conditionsof strong biological heat productionsuch as that occurring with modern highcell density processes.

All wetted materials of the BioBLU 1c/fsingle-use bioreactors are certified»USP Class VI« and are in compliancewith US-FDA requirements.

More information atwww.eppendorf.com/bioblu

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.

BioBLU 1c for cell cul ture BioBLU 1f f or microbiolog y

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13A REALLY COOL PRODUCT FAMILY: NEW BRUNSWICK™ ULT FREEZERS · STRAIGHT FROM THE LAB

A Really Cool Product Family:

New Brunswick™ ULT Freezers

MARY LISA SASSANO, EPPENDORF, INC., ENFIELD, CT, USA

Impetus to energy-efficiency

New Brunswick ULT freezers are well-known for their energy-efficient perfor-mance. Innova® freezers provide up to30 % more storage capacity comparedto traditionally insulated freezers byusing advanced vacuum insulated paneltechnology which allows a reduction infreezer wall thickness. »Premium« freezersoffer the same top-quality, but use conven-tional foam insulation for a cost-savingalternative.

High Efficiency HEF® and Green »G«freezers further reduce energy con-sumption, making them among the mostenergy-efficient in the market. HEFfreezers, for example, reduce energyconsumption by up to 59 % when com-pared to competitive models.

Vacuum insulation panels combinedwith traditional polyurethane foamform a 130 mm thick layer of insulation.These freezers, in addition, feature apowerful, low-noise condenser fan andcompressor system thus providingexceptional energy efficiency. The 50 Hzversions of HEF and Green modelsadditionally use environmentally-safe

hydrocarbon-based refrigerants tofurther reduce energy consumption andgreenhouse gases.

Smart features

All models include ergonomically posi-tioned easy-open doors with a moldedhandle, adjustable height shelves, lownoise and low heat output. A bright LEDcontrol panel mounted at eye level onuprights is easy-to-read, and flush

mounted for easy wipe-down. A wash-able filter is front mounted and easilyaccessed without tools. Also, a heatedair vent with ice-cleaning plunger pre-vents vacuum formation, allowing thedoor to be quickly opened throughoutthe day. These ULT freezers are de-signed to fit through standard doorwaysand elevators, allowing for ease of

movement.

Tip: The TCA-3 Temperature MonitoringSystem offers added security. Thisoptional unit combines an independenttemperature monitor with an alarm,electronic chart recorder, and an auto-dialer into one small pod. The TCA-3provides users a considerable measureof added security for their sample pro-tection.

The current Eppendorf line of New Brunswick™ Ultra-Low Temperature (ULT) freezers has become so com-

prehensive 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 101 – 760 L capacities.

Innova Freezersreduce insulating wallthickness to provide

up to 30% morestorage capacity

HEF Freezersimprove energy

efficiency by addingvacuum insulation

panels to traditionalfoam in a 130 mm

space

<−− 80 mm −−>

Premiumwalls

Innovawalls

HEFwalls

<−−−−− 130 mm −−−−−> <−−−−− 130 mm −−−−−>

Polyurethane Foam Vacuum Insulation Panels

There is a New Brunswick freezer perfectly suited for every lab! New Brunswick™ HEF® Freezers • Ref.no. 267

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SERVICE · PRIZE COMPETITION4

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 thelight grey boxes of the crossword in the correct order. Send usthe solution until 30 th June 2014.

1 2 3 4 5 6 7 8 9 10 11

12 13

14 15 16

17 18 19

20 21

22 23 24 25

26 27 28

29 30 31 32

33 34 35 36 37

38 39 40

41 42 43 44

45 46 47

48

You can either use the reply fax (p. 15), send us an e-mailto [email protected], or participate online atwww.eppendorf.com/bn-service.

All correct answers will be considered for a prize. Winners willbe 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. 42.

ACROSS

1 Centrigrade temperature scale7 These are a-changin (according to

Bob D.)12 It's fun to stay there13 Opposite of yes14 Computer-aided design (abbrev.)15 Popular lab instrument17 Chemical symbol for lithium18 Human immunodeficiency virus

(abbrev.)19 Word expressing approval or assent

(abbrev.)20 Spanish definite article21 ISO code of Estonia22 Warning signal24 Ribonucleic acid (abbrev.)26 Professional Golfers Association

(abbrev.)27 Male given name

28 ISO code of Turkey29 ISO code of Nauru30 Not fake or false (e.g., in Madrid)31 ISO code of Greenland33 The B in BMI35 Capital of Norway38 Number prefix denoting two40 Moon of saturn41 Greek prefix for young, new42 Object detection system based on

radio waves45 Founder of an annual international

awards bestowed in a number ofcategories (first name)

46 Chemical symbol for silicon48 Biotechnological process

DOWN

1 Device used for PCR2 Electronic message3 Flat panel display (abbrev.)4 ISO code of Saudi-Arabia5 Comprehensive broad and versatile6 Morse code distress signal7 Travel by walking8 Male given name9 Part of the name of Eppendorf

thermocyclers10 A particular part of something11 Severe acute respiratory syndrome

(abbrev.)15 Princess of Wales † (short name)16 Flows through Turin and Piacenza18 Female given Name22 Chemical symbol for silver23 To sell or write25 Noble gas

26 Thin, long instrument used forexploration

29 nota bene (abbrev.)32 French law34 Electronic component36 Completes Lucia, Louis and Tropez37 Tool of the American cowboy39 Rail vehicle (e. g., in Vienna, Austria)41 10-9 m (abbrev.)43 Typical British drink (produced using

48 across)44 The A in MOMA47 Chemical symbol for indium

Amazon® is a registered trademark of Amazon.com, Inc., USA. DASGIP® is a registeredtrademark of DASGIP Information and Process Technology GmbH, Germany. HEF® andInnova® are registered trademarks of New Brunswick Scientific Co. Inc, USA. Hellma® is aregistered trademark of Hellma GmbH & Co. KG, Germany. Life Technologies® is a registeredtrademark of Life Technologies Corp., USA. Science® is a registered trademark of American

Association for the Advancement of Science, USA. UltraPure

™

is a trademark of LifeTechnologies Corp., USA . YouTube® is a registered trademark of Google Inc. Corp., USA.

Disclaimer and Trademarks Information

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®, EppendorfReference®, 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 NewBrunswick™ logo are trademarks of Eppendorf AG, Germany. Errors and omissions excepted. Copyright © January 2014.

Prize Competition

1st

Prize:1 Multipette® M4* with2 sets of Combitips advanced® of your choice*(US/CAN: Repeater® M4)

2nd to 5 th Prize:1 Amazon® Voucherworth 50.– Euros

6th to 15 th Prize:200 bonus ep-points® each

8/12/2019 BN40_IN_150dpi


Please send me information on the following products: Kindly help us to update your subscription details:

253 New Brunswick™ S41i CO2 Incubator Shaker Please change my details (see above).

261 Eppendorf PlateReader AF2200 I am a new subscriber! Please send meEppendorf BioNews on a regular basis(2 issues per year, free of charge).262 ep Dualfilter T.I.P.S.® SealMax

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

15REPLY FAX / READERS' SERVICE · SERVICE

Solution of BioNews No. 40 prize competition:

Visit www.eppendorf.com/bn-service to subscribe or to request literature online!

To Eppendorf AG, Hamburg, Germany, Attn. Carolyn Taubert, Fax (+49) 40 - 5 38 01- 8 40(Please – print or type, no private address)

Company/ University / Clinic Title / Name

Institute Address

Department / Function Address

E-Mail Telephone

Closing date: 30 th June 2014 Visit www.eppendorf.com/bn-service to participate online.

F E E E T

8/12/2019 BN40_IN_150dpi


There’s only oneGalileo Galilei

For your career in science, there’s only one

Careers

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.

B

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