SSUE INTER vwr

Studying living cellsindividually

page 10

Tools to study cellular signaltransduction

page 6

ISSUE 19 - WINTER 2007The Market Source for Life Science

vwr.com

VWR Collection geldocumentation system

page 32

VWRBiosciencessection – Genomicsround-up

page 45

Are you registered for our life science e-newsletter? Get all the latest news and offers, go to www.vwr.com..Find bioMarke there too with lots more information and downloads.

bioMarkeMagazine-19.qxp 10/10/2007 16:03 Page 1

Abgene*

Air Liquide

AppliChem

Axygen

BD Biosciences

Binder

Brand

BTX Harvard Apparatus

CBS

Merck Biosciences

Molecular BioProducts

Nalgene

Nunc

Omega Bio-Tek

Operon

Pall Life Sciences

5 PRIME

Sartorius

Spectrum

Thermo Scientific

Whatman

Wheaton

ABgene* products are not availablein Italy, Belgium and the Netherlands

table of contentsProteins

Merck - ProteoExtract® Subcellular Proteome Extraction Kits 3

Pall Life Sciences - Increased productivity using Pall’s MinimateTM Capsules 4

Whatman - Tools to study cellular signal transduction 6

VWR Collection - Trends in cryopreservation 8

NUNCTM LiveCell ArrayTM - Study living cells individually 10

Cell biology

BD Biosciences - The same tube with a NEW innovative cap design… 14

Applichem - Recognise and avoid interference in Immunoassays 16

Air Liquide - Arpege Gas: an new range of cryogenic containers 18

Thermo Scientific - Varioskan® Flash and MicrotiterTM plates for photometry 20

Molecular biology

5-PRIME - Genomic DNA purification tools 22

Thermo Scientific ABgene® SuperPlateTM - Peace of mind in a warped world! 24

BTX - High efficiency transfection of primary human & mouse T lymphocytes 26

CBS Scientific - Protection against in sensitive PCR amplification reactions 28

Omega bio-tek - E.Z.N.A.® Fastfilter Plasmid Midi and Mini kits 30

VWR Collection -New gel documentation system 32

Axygen - Plates and mats for Real-Time PCR and sequencing 34

Sample preparation

Spectrum - MediaKap® hollow fiber media filters for sterile filtration 36

Binder - Standards compliant hot air sterilisation at 180 °C 12

Liquid handling

MBP - ART® and HLTTM Gel Tips 40

Brand - Transferpette® S - now also available as multichannel pipettes! 38

Wheaton - Preparation of agar plates with antibiotics using Omnispense® Plus 42

Biosciences

Thermo Scientific - New! ProjectPeptideTM Service 44

VWR Biosciences - Genomics roundup 45

VWR I n t e r n a t i o n a l Issue 19 N o v e m b e r 2 0 0 72

editorialAs the days grow shorter and the temperatures drop, VWRbioMarke wintermagazine is here to warm up the brain at least! Another super size issue packedwith technical data and application stories from cell biology to liquid handling withmolecular biology and proteins in between. We are particularly proud of the newVWR Collection gel documentation system that has been launched to market praisefor it’s solid construction, top performance and a price that will not break the bank.

Check out our promotional flyer the VWRbioMarke shop that includes a widevariety of offers and saving on hundreds of products.

Very best regards for a Merry Christmas and happy, healthy and prosperous 2008.

The VWRbioMarke Team

PS You will also be able to find on the web with additional downloads and otherinformation where available. To make sure that you get all the news about theseand other new items as well as special offers and new catalogues register on ourwebsite to get the VWR e-newsletter.

EditorVWR International Europe bvbaHaasrode Researchpark Zone 3Geldenaaksebaan 4643001 LeuvenBelgium

CopywritingVWR International Europe bvba

Layout and typesettingMarketing Services VWR

PrintingStork, GermanyNo part of this publication may be reproduced or copiedwithout prior permission by writing of VWR InternationalEurope.

Run77.650 copiesPublication date: November 2007

Due to the high sales volume of promoted articles someitems may be temporarily out of stock - VWR Terms andConditions of Sale apply.


VWR I n t e r n a t i o n a l Issue 19 N o v e m b e r 2 0 0 7 3

For more information on these products contact your local VWR sales office,send an e-mail to [email protected] or visit our website www.vwr.com

ProteoExtract® Subcellular ProteomeExtraction Kit

Proteins

Free timerwith every kit while stocks last

See our promotion in the

Shop!

Description Pack Cat. No.

ProteoExtract® Subcellular Proteome Extraction Kit 20 rxn 539790-1ProteoExtract® Subcellular Proteome Extraction Kit, Mini 10 rxn 539791-1

For full technical details of these, and other, sample preparation kits for protein research, please contact your local VWR sales specialist.

PRODUCT FEATURESKit configuration All reagents included: extraction & wash buffers,

protease inhibitor cocktail, Benzonase® nuclease

Kit sizes For 10 or 20 samples

Sample type Mammalian adherent and suspension - growncells, mammalian tissue

Sample volume 106-107 cells, 25-50 mg tissue, scalable

Fractions 4 fractions: cytosolic, membrane/organelles,nuclear, and cytoskeletal

Downstream applications 1-& 2-D-GE, ELISA, SELDI, immunoblotting,activity assays, subcellular redistribution assays,arrays

The ProteoExtract® Subcellular Proteome Extraction Kit (S-PEK) is designed for reproducibleextraction of subcellular proteomes from mammalian cells. Based on different solubilities ofcertain subcellular compartments the S-PEK uses proprietary chemistries to yield four sub-proteome fractions that are enriched in cytosolic, membrane/organelle, nuclear, andcytoskeletal proteins. In the case of adherent cells, the procedure is performed directly in thetissue culture dish without the need for cell removal. For suspension grown cells, extractionstarts with gentle sedimentation and washing of cells. For tissues, the isolation of viable cellsis required before proceeding with the extraction protocol. The especially mild S-PEK procedureresults in non-denatured proteins suitable for many downstream applications.

Analysis of protein distribution profiles to characterisecellular changes, exemplified by a time-course analysisof NFκκB redistribution in stimulated cells.

A431 cells were stimulated with 0,2 µg/ml TNFα, and stepwiseextraction of cytosolic fraction (F1), organelle/membranefraction (F2) nuclear fraction (F3) and cytoskeletal proteins(F4) was performed using the S-PEK procedure. Cell fractionswere separated by SDS-PAGE and blotted onto nitrocellulosemembrane. The membrane was probed with anti-NFκB. Thetime-course analysis demonstrates a measurable translocationof NFκB from the cytoplasm to the nucleus as early as 5 minand a stronger response at 15 min.

APPLICATIONSS-PEK fractions are particularly well suited forsensitive proteomics applications:

• Subcellular redistribution assays to monitorprotein translocation

• Enzyme activity assays including reportergene assays and kinase assays

• SELDI-profiling

• Non-denaturing gel electrophoresis

• Assaying protein expression levels usingfluorescent labelled subcellular extracts inmicroarrays

Redistribution of NFκκB upon stimulation of cells


Ultrafiltration preserves biologicalactivity and timeUltrafiltration (UF) techniques rely on the useof fluid pressure to drive the migration of thesmaller molecules through a UF membranewith the simultaneous retention of largermolecules. While chemical precipitation can beused to concentrate a protein sample,separation with ultrafiltration is based onmechanical rather than chemical interactionsallowing a researcher to perform sampleconcentration without the addition ofdenaturing solvents or salts. Buffer exchangeusing dialysis technologies uses large volumesof buffer and, since the only force acting uponthe solution is diffusion, the process can takeseveral days. Pre-assembled and simple-to-useultrafiltration devices can rapidly performeither concentration or buffer exchangeprocedures without the extensive handlingrequired for many other techniques.

Ultrafiltration can be performed in one of twooperational modes: Direct Flow Filtration(DFF), or Tangential Flow Filtration (TFF) (Figure 1). DFF works well for small volumes(up to 30 ml) using centrifugal devices,however, DFF technologies can fall prey toproblems with membrane fouling. To reducethe formation of a gel layer, cross flow can begenerated on the upstream side of themembrane using a floating stir barconfiguration (stirred cell) or by creating acontrolled laminar flow. While stirred celloperations tend to improve UF performance,they are still limited in achieving the optimalperformance since the velocity andsubsequent level of agitation is dependent onthe sweep of the bar that varies along theradius of the sweep.

Reduce processing time using theMinimate™ capsule

TFF operations allow a uniform and gentlerecirculation of sample flow over the surfaceof a membrane effectively controllingmembrane fouling. Use of the entiremembrane surface results in improved fluxrates significantly reducing processing timesand increasing productivity. The MinimateTM

capsule is pre-assembled, ready to use andcan process volumes up to 1 litre without theneed for user intervention. In contrast, moststirred-cell devices have a 350 ml or smallerprocessing capacity and must be openedrepeatedly, risking integrity failures andrequiring valuable technician time.

Processing time is a function of overall deviceperformance. The processing operation of five

MinimateTM 10 K OmegaTM capsules wasdirectly compared to similar processingconditions using comparable stirred cellconfigurations containing high-flow PES orregenerated cellulose membranes.A 2 mg/ml BSA solution (1 litre) wasconcentrated ten-fold (to 100 ml) using theMinimateTM capsule to achieve concentrationin just over an hour (Figure 2). In contrast,processing times for stirred cell devices tookfive times longer regardless of membraneconfiguration and required the user to refillthe device repeatedly.


Increased productivity using Pall’s MinimateTM capsules Kevin Seeley, Ph.D. Scientific and Laboratory Services, Pall Corporation

(A) Sample solution flows through the feedchannel and along (tangent to) the surface of themembrane as well as through the membrane. Thecrossflow prevents build up of molecules at thesurface that can cause fouling.(B) The TFF process prevents the rapid decline influx rate seen in direct flow filtration allowing agreater volume to be processed per unit area ofmembrane surface.

(A) The feed is directed into the membrane.Molecules larger than the pores accumulate atthe membrane surface to form a gel, which foulsthe surface, blocking the flow of liquid throughthe membrane. (B) As the volume filtered increases, foulingincreases and the flux rate decreases rapidly.

(A) (B)

(A) (B)

Figure 1. Graphic comparison of DFF versus TFF

Figure 2. Use of Minimate capsulesignificantly reduces processing times A 2 mg/ml BSA solution was concentrated tenfold(1000 to 100 ml) in either a 350 ml stirred celldevice or Minimate capsule. The Minimatecapsule contained a pre-assembled Omega 10 Kmembrane sandwich with a cross flow set at 50 ml/min. with retentate loop backpressureapplied to create an initial filtrate flow of about15 ml/min. The stirred cell devices usedpolyethersulfone (PES) or regenerated cellulose(RC) disks and were pressurised with filtered airat 55 psi giving a starting filtrate flow of about 6 ml/min. Error bars indicate standard error forfive independent runs.

Diagram 1 Minimate TFF capsule systemwith pump, pressure gauge,retentate screw clamp,reservoirs and tubingconnections



For more information on these products contact your local VWR sales office,send an e-mail to [email protected] or visit our website www.vwr.comProteins

Handling and device integrity

Integrity failures during processing not onlyresult in lost time, but also end up with thedestruction of valuable sample. TheMinimateTM capsule is manufactured as asealed, self-contained capsule that requires noend-user assembly. Using TFF, a 1 litre proteinsample can be processed in a single stepreducing the risk of disturbing the sample. Incontrast, the SC devices have a complexassembly that requires the UF membranedisks to be placed on a device landing and

screwed into place, an action that can createleaks or bypass. By monitoring the filtrateprotein bypass, we observed that thecompetitive PES membrane types sufferedsignificant integrity failures in stirred celloperations (Figure 3). The MinimateTM andregenerated cellulose devices were allintegral, however, the competitive PESmembranes failed in 5 out of 10 runs each atdifferent stages in the processing.

Purification of cyclooxygenase-2(COX-2)

A study was performed to validate the use of the MinimateTM over the stirred cellconfiguration for the purification of the ovineCOX-2 enzyme from tissue homogenate (Table1). In the study, they were able to easilydevelop a new protocol that replaced thestirred-cell device in an enzyme productionprocess. They found that the COX-2 specificactivity derived from the TFF procedurematched that from the stirred cell device theyhad been using. Most critical however, was

that the processing time was cut to nearly athird of the time for TFF ultimately allowing asecond run to be performed during a singleshift.

Conclusions

The simple-to-operate MinimateTM TFF capsuleis the most rapid, reliable choice for bufferexchange or protein concentration for samplesup to 1 litre. In contrast, stirred-cell systemsrequire assembly, operate more slowly, requiremore intensive monitoring, and have to befilled multiple times in order to processsample volumes up to 1 litre. While theMinimateTM TFF capsule is a valuable tool forsmall process volumes (<1 l), larger volumescan be processed by multiplexing severalMinimateTM TFF capsules in a parallelconfiguration. In addition, the MinimateTM TFFcapsule was designed with the same flowpath length as larger TFF devices, savingvaluable optimisation time when scalingbeyond lab scale to volumes used in pilot andproduction plants.

When you find a product that dramatically speeds the discoveryprocess, you get more time for what you really want to do - discover.Pall’s MinimateTM tangential flow filtration capsule allows forconcentration or diafiltration (desalting) of biomolecules.Economically designed to be disposable, it lets researchers eliminatethe cleaning and cleaning validation steps. However, for non-criticalapplications, the MinimateTM capsule can easily be cleaned and re-used. Developed in response to real laboratory needs like yours,the MinimateTM is scalable, provides high product recovery, and is100% integrity tested to ensure reliable performance.So the next generation of biopharmaceuticals gets to those whoneed them sooner. After all, the faster your discovery moves throughthe pipeline, the more time people have to benefit from it.

Pall – Making time for discovery

Table 1 Stirred cell MinimateIndependent purification 1 2 3 1 2 3

Initial protein conc. (mg/ml) 15,0 14,5 15,0 13,6 14,1 14,7 Initial specific activity (µ/mg) 66,7 62,1 70,9 73,5 67,4 74,8 Concentration factor 50 46 50 40 44 41Final specific activity 220 196 191 200 217 206 Final yield (SA/mg total protein) 0,048 0,045 0,043 0,042 0,044 0,040Time to process (hr) 8,0 8,0 8,0 2,5 2,5 2,5

Figure 3. The Pre-assembled Minimateeliminates integrity failures Sample concentration was performed asdescribed in Fig.2. Aliquots of the startingmaterial, final retentate, and final filtrate poolswere analysed for protein concentration at 280nm. Average concentrations are plotted witherror bars indicating standard error for (5 - Minimate), (10 - SC PES), and (5 - SC RC) runsrespectively. For simplicity in the graphicalrepresentation, the retentate values are plottedon the Y2 axis to accommodate the ten-foldconcentration that occurred in the processing.

Description Cat. No.

MinimateTM TFF 650D OmegaTM 1/pk 515-0124MinimateTM TFF 1K OmegaTM 1/pk 515-0116MinimateTM TFF 3K OmegaTM 1/pk 515-0117MinimateTM TFF 5K OmegaTM 1/pk 515-0118MinimateTM TFF 10K OmegaTM 1/pk 516-6879MinimateTM TFF 30K OmegaTM 1/pk 516-6880


MinimateTM TFF 50K OmegaTM 1/pk 515-0119MinimateTM TFF 70K OmegaTM 1/pk 515-0120MinimateTM TFF 100K OmegaTM 1/pk 516-6881MinimateTM TFF 300K OmegaTM 1/pk 515-0121MinimateTM TFF 500K OmegaTM 1/pk 515-0122MinimateTM TFF 1000K OmegaTM 1/pk 515-0123


Proteins interact in a multitude of

different ways. When a protein

kinase phosphorylates a target

molecule, these two proteins

certainly interact; however, the

term protein-protein interaction

usually describes binding of a

noncatalytic domain of one protein

to a specific motif in a second

protein. Most interaction domains

are small, globular entities which

range in size from about 35 to 150

amino acids. Their binding partners

are usually very small motifs of less

than 10 amino acids. Protein

domains are modular entities

embedded in larger protein

sequences. Their function is to

tether together multiprotein

complexes. Recruitment of proteins

into signal transduction cascades

via protein interaction domains is a

general principle found in many

receptor mediated pathways.

In many cases, binding is regulated by post-translational modification of the motif,such as acetylation or phosphorylation. Infact, one of the most important functions ofprotein phosphorylation is to generate orabolish binding sites. Protein domains can begrouped by sequence homologies intofamilies such as SH2 and SH3 (Src-Homology2 and 3) or WW (a domain with twoconserved tryptophans). These families canbe grouped by their target motifs intophospho-Tyr binding domains (SH2, PTB),phospo-Ser/Thr binding (eg WW, 14-3-3),proline-rich motif binding (eg SH3) etc.

A widely used method to screen for protein-protein interaction is the yeast two-hybrid system. Tandem affinitypurification (tap-tagging) of proteincomplexes followed by identification of the

proteins by Mass Spectrometry (MS) is analternative approach. Currently, lack ofconsistency between these methods, as wellas within the same method, is a seriousdrawback.

Protein-protein interactiondata obtained frommicroarray experiments onFAST Slides is reliable

Several of the well-known interactions ofcalmodulin were missed in the large-scaleyeast two-hybrid and/or affinity purification-MS studies, including the interaction ofcalmodulin with calmodulin kinase(Schweitzer et al. 2003). In order todemonstrate that a protein array can identifyreciprocal interaction of proteins, Schweitzeret al. (2003) probed identical yeast whole-proteome arrays with biotinylatedcalmodulin and biotinylated calmodulinkinase, respectively. Detection was madewith Cy5-labelled streptavidin. Theinteraction was found irrespective of whichprotein was present in solution and whichwas immobilised on the FAST Slide surface.

Seitz et al. (2006) used protein microarrayson FAST Slides and Biacore technology as


Protein microarrays on Whatman FAST® Slides: tools tostudy cellular signal transductionPart 2: studying protein-protein interactions



For more information on these products contact your local VWR sales office,send an e-mail to [email protected] or visit our website www.vwr.comProteins

two independent methods to verify putativeinteraction partners of two members of theS100 protein family, S100A6 (Calcyclin) andS100B. S100 family proteins arecharacterised by two EFh (EF-hand) Ca2+

binding domains, which undergo a majorconformational change upon binding of adivalent cation. This conformational changeexposes a hydrophobic target binding sitewhich can interact with effector proteins,thus eliciting a physiological response. 80recombinant human proteins (RGS-His6-tagged) derived from a proteome-wideexpression library were arrayed. The twoS100 proteins, S100A6 and S100B, wereexpressed and purified as GST fusionproteins. Their affinity to the arrayed “preyproteins” was assayed in the presence ofcalcium ions and, as a control, in thepresence of EDTA without Ca2+. An anti-GST-Cy5 conjugate was used for detection. Thesame set of proteins was analysed usingBiacore technology. For S100A6, 9 and 11interactions were found using microarray andBiacore, respectively. 6 of these wereidentical with both methods. Interestingly,there was less overlap in the Biacore analysiswhen the reverse experiments wereperformed (ie probing of immobilised S100proteins with the “prey protein” in thesolution phase).

Protein-domain microarraysidentify novel protein-protein interactions

A microarray displaying protein interactiondomains is a valuable tool to study theprotein-protein interaction network of thecell. Espejo et al. (2002) expressed 145proteins containing interaction domains asGST fusion proteins (33 WW, 23 SH3, 17 SH2,23 PH, 23 PDZ, seven 14-3-3, 5 PTB, 4 FHA,8 FF and 2 KH). These, plus 67 proteins “of general interest” but without canonicalprotein interaction domains, were arrayed onFAST Slides. As a test for specificity of thischip, several binding motifs were synthesisedas biotinylated peptides. Motifs included theproline-rich P3 motif of Sam68 (which binds

SH3 and WW domains) and theproline/glycine/methionine (PGM) motif ofthe ribonucleoprotein SmB’ (which binds to aspecific subfamily of WW domains). Thesepeptides, pre-bound to streptavidin-Cy3 orstreptavidin-Cy5, were subsequently used toprobe the array. Unique binding profiles werefound for each of the peptides. Interactionwith the arrayed proteins was as predictedfrom the presence of domains.

Arginine-methylation is known to reducebinding of the P3 motif of Sam68 to SH3 butnot to WW domains. To test this, a mixture ofunmethylated peptide conjugated tostreptavidin-Cy3 and methylated peptideconjugated to streptavidin-Cy5 was used toprobe one chip. The unmethylated peptidewas found to bind two WW domains and sixSH3 domains. The methylated P3 bound thesame two WW but only three SH3 domains.The next step was to analyse the interactionof Sam68 (Src-associated during mitosis 68)and the splicing factor SmB’ from cell lysateswith the arrayed domains. The proline-richmotifs (P3 and PGM) are different and asexpected, the binding patterns on the chipwere distinct, with some overlap. Bindingpatterns of both proteins were very similar tothose observed with the synthetic bindingmotifs.

The same protein domain array was used tostudy the function of the tumour suppressorprotein tuberin (Liu et al. 2002). Tuberin is atarget for phosphorylation by Akt. It plays arole in receptor tyrosine kinase signalling bynegatively regulating phosphatidylinositol 3’ kinase (PI3K) downstream of Akt. An in-silico analysis of tuberin indicated severalhigh-stringency 14-3-3 binding motifs which

overlap with Akt phosphorylation sites. Shortpeptides containing predicted 14-3-3 bindingmotifs were synthesised and fluorophore-tagged. One peptide harbouring the Ser939

residue was found to have no specificbinding to 14-3-3 domains on the array in itsunphosphorylated form. However, thephosphorylated form of the peptide did bindto 14-3-3α and, to a lesser extent, to 14-3-3ζ domains. To confirm this finding in vivo,all 14-3-3 isoforms were expressed as GSTfusion proteins and incubated with lysatesfrom serum-stimulated NIH3T3 cells. GSTpull-down experiments were performed, andtuberin was found to co-precipitate with all14-3-3 isoforms. In the presence of thephosphorylated Ser939 peptide this tuberin co-precipitation was greatly reduced. Theseresults corroborate the domain array data.A very comprehensive description ofgenerating and processing proteininteraction domain arrays can be found inEspejo and Bedford (2003).

References1. Espejo et al. 2002. A protein-domain microarray

identifies novel protein-protein interactions.Biochem J. 367(3): 697-702.

2. Espejo and Bedford 2004. Protein-domainmicroarrays. Methods in Molecular Biology. Vol264: 173-181.

3. Liu et al. 2002. 14-3-3 Interacts with the tumoursuppressor tuberin at Akt phosphorylation site(s).Cancer Research 62: 6475-6480.

4. Schweitzer et al. 2003. Microarrays tocharacterise protein interactions on a whole-proteome scale. Proteomics 3: 2190-2199.

5. Seitz et al. 2006. Differential binding studiesapplying functional protein microarrays andsurface plasmon resonance. Proteomics 6: 5132-5139.

Contributing AuthorsDr Michael WaltherDr Betsy MoranDr Michael HarveyDr Breck ParkerLisa ML D’Anzi

EditorsSenior Editor: Betsy A Moran, PhDManaging Editor: Lisa ML D’Anzi

Interaction of a protein in solution witharrayed proteins can also be detectedutilising antibodies specific for thesolution-phase protein.


Cryopreservation

For cells or whole tissues preserved bycooling down to cryogenic temperatures, anybiological activity is effectively stopped. If thematerial is not sufficiently protected againstextracellular ice formation, dehydration andintracellular ice formation, it is oftendamaged during the freezing process and/orduring the thawing process, compromisingviability. Cryoprotectants, rate freezing,vitrification and controlled thawing can bethe means or mechanisms to reduce thefreezing damage.

Liquid Nitrogen (LN2) is a relativelyinexpensive cryogenic liquid and is for thisreason widely used for different cryogenicpurposes. Liquifying at –196 °C and freezingat –210 °C LN2 is inert, colourless,non-corrosive, non flammable and very smallamounts vapourise into large amounts of gas.

Liquid Storage

Since the invention of the vacuum Dewar in1892 it has been widely used for long termstorage in LN2 - despite submersion andretrieval being a risky and unpleasant activity.For many years this was the only availablemethod to store material at a temperaturewell below the re-crystallisation point ofwater where ice crystals could causeirreparable damage to the stored material.

Like all liquids, LN2 is a possible transporter ofcontaminants and ice sedimentsaccumulating in storage Dewars can becomethe source of microbial contamination ofstored samples. There has been cleardemonstration of the transmission ofHepatitis B between frozen bone marrowsamples in a liquid nitrogen storage tank,which resulted in clinical infection ontransplantation into patients (Teddar et al.,1995; Fountain et al., 1997).

Whilst aseptic techniques are the basis ofsafeguarding the samples in storage, thepossible leakage of contaminated LN2 intosubmerged samples is often overlooked or

underestimated. So even though modernstorage vials when used correctly are leakproof, manufacturers will demand that theirvials are not submerged as they have nocontrol over the handling of the cap-vialconnection.

Cryogenic temperatures are certainly hard onthe plastic vials and caps with perhaps thebiggest threat coming from the differentialpressure that is developed when the vials arecooled. The state of an amount of gas isdetermined by its pressure, volume andtemperature, according to the equation:pV=nRT. When submerging into LiquidNitrogen reduces the temperature, thepressure inside the vial will reduce as well,creating a difference in pressure. This forcesLN2 into the vial if the cap is not properlyscrewed on, has a deformed gasket or isscrewed on too tight. As well as the cross-contamination risk, the ingress of LN2 into thevial could possibly cause an explosion whenthe vial is taken into the lab environment, dueto the rapid boil off of the trapped nitrogenwhen LN2 vapourises into N2 it expands 700times.

Vapour Phase Storage

When in the 1980’s HIV/AIDS was firstrecognised, the possible cross-contaminationrisk of storing samples in LN2 became an


Developments in cryogenic storageR.V.D.BoschCryogenic Industry Consultant

Cryogenics developed in the 19th

century as a result of efforts by

scientists to liquify permanent

gases. Oxygen and Nitrogen, as the

primary constituents of air,

received the most attention. James

Dewar (1842-1923) played a key

role inventing the first insulating

bottle for the storage and

transport of liquid gases. Dewars

are still widely used today.

James Dewar

Traditional vapour phase unit Gas vs Liquid storage


increasing concern. Bielanski, et al., (2000)showed that when LN2 is contaminated witha virus, embryos stored in unsealed containerscan become contaminated.

Storing samples in the vapour of LN2

eliminates the risk of cross-contaminationthrough LN2. This way of storage is mostlyperformed in the same refrigerators (Dewartype or autofill type) that are used forordinary liquid storage but the liquid level isreduced to 1/3 and the racks with thesamples are either separated from the liquidby the use of a platform, or the lowestpositions of the racks (the positions thatstand in the liquid) are not used for samplestorage.

This solution however reduces storagecapacity, and produces unpredictable vapourtemperatures and temperature gradientsthroughout the vessel. Unless biologicalmaterial is stored at temperatures below theglass transition of aqueous solutions (approx.-130 to -140 °C; Mazur, 1970), the viability

decreases during long term storage. Inaddition, some materials such as Hybridomasare very sensitive to temperature changesbetween -130 and -190 °C.

Conventional vapour phase freezers cannotmeet these ideal requirements and althoughsome products are retrofitted with heatshunts to increase the evaporation of LN2,they still do not reach the stability andtemperature that submersion provides.

Latest Developments -190 °C Dry Storage

It was in the year 2000 that an Americanmanufacturer (Custom Biogenic Systems) ofCryogenic Storage Freezers launched the first-190 °C Dry Storage system - the first majorinnovation in cryogenic storage since JamesDewar.

This new storage system uses the samevacuum vessel as conventional freezers, butinstead of having the pool of LN2 in the tank,it is in the walls of the storage freezer.

Hence there can be no risk of cross-contamination through LN2 and the aircirculation in the freezer in combination withthe convection from the bottom and the wallsprovides a very stabile temperature of about -190 °C from top to bottom. Plus the entireinterior of the vessel can be used for storageincreasing capacity.

The VWR Collection personal freezer based onthe dry shipper principle has a LN2 pump thatadds LN2 to the absorbant as the LN2 isevaporated. The system provides a dry, stable-190 °C environment for storing tissues andcells and as it doesn’t require a pressurisedLN2 supply is safe to be used at the bench.

European Directives

The risks of cross-contamination, as well asthe risks of viability reduction by large verticaltemperature gradients during cryogenicstorage, are a serious concern to all European

governments prompting Directive2004/23/EC, setting standards of quality andsafety for the donation, procurement, testing,processing, preservation, storage anddistribution of human tissues and cells.Specifically:• Article 20.3 of this Directive addresses the

fact that establishments shall include intheir standard operating proceduresspecial provisions in order to prevent thecontamination of other tissues or cells.

• Article 21.3 of this Directive addresses thefact that establishments shall establishand apply procedures that prevent anysituation arising that might adverselyaffect the functioning or integrity oftissues and cells.

So if your human tissues and cells are stillsubmerged in LN2 or you suspect temperaturefluctuations or gradients in the vessel it mightbe time to reconsider the methodology.

References1. Tedder et al 1995

Hepatitis B transmission from contaminatedcryopreservation tankLancet 346 : 137-140

2. Fountain et al 1997Liquid Nitrogen freezer a potential source ofmicrobial contamination of Hematopoietic stemcell componentsTransfusion 37 : 585-591

3. Bielanski et al 2000Viral contamination of embryos cryopreserved inLiquid Nitrogen. Cryobiology 40 : 110-116


For more information on these products contact your local VWR sales office,send an e-mail to [email protected] or visit our website www.vwr.comCell biology

Cryogenics



NUNC™ LiveCell Array™ Study living cells individually

• First slide-based tool for real-timestudy of individual, living cells(adhering or non-adhering) withinheterogeneous populations

• Disposable, sterile microscopeslide with an embeddedtransparent array of picowells

• Enables multiple functional assayson living cells followed by post-fixation studies on the samecells

• Staining, rinsing and perfusion donot displace cells

• Compatible with standardmicroscopes

• Image Analysis Software,commercially available, candesignate an address to each cell

LiveCell™ is a licensed trademark of MolecularCytomics Inc.

The Premier High Content Analysis Tool for Cell Biologists

Unique capabilities of the NUNC LiveCell ArrayTM with individual,living non-adherent cells:

NUNC LiveCell ArrayTM Flow Cytometry Microscopy

Fluid exchange w/o cell displacement Yes No No

Minimal reagent volume Yes No No

Minimal sample size Yes No Some

Non-stressed cell environment Yes No Some

Monolayer cell distribution Yes No No

Real-time kinetics Yes No No

Multiplexing: Correlation of pre- and post-fixation assays on the same cells Yes No No

Maximise utilisation ofvaluable cellularsamples by performingmultiple functional assayson living cells followed bypost-fixation studies on thesame cells

Study heterogeneousprimary cell populationssuch as bone marrow,blood, beta and sperm cells,observing individualresponses to treatmentsand identifying rare cells(eg. stem cells)

Perform kineticmeasurements of non-synchronous activities inindividual cells, observingeach cell at its own pace

The NUNCLiveCell Arraymicroscope slideis currentlyavailable in fourconfigurations:

Time lapse imaging of changes in mitochondrialmembrane potential in individual non-adherent cells

The Individual Cell Array with15-micron or 20-micron wells,designed for primary bloodcells, non-adhering cell lines,primary bone marrow cellsand beta cells

The Multi-Cell Array with 100-micron or 250-micronwells, designed for large cellssuch as oocytes, eggs, plantcells, and neuronal cells, smallgroups of cells, cell clustersand tissue-like cell aggregates

Bone marrow cells and lymph node cells

Functional measurements on living cells; post-fixation chromatic staining on the same cells


• OptiCellTM is a unique cell culture format for growing, monitoring,and transporting cells

• Two parallel gas-permeable, cell culture treated polystyrenemembranes attached to a standard microwell plate-sized frame- Each side has a growth area of 50 cm², total 100 cm²- 75 µm thick membranes, 2 mm apart

• The OptiCellTM system allows gaseous exchange of both oxygenand carbon dioxide through the approx. 100 µm polystyrenemembranes. The membrane has a low permeability to watervapour so evaporation is minimal

• Thin profile design maximises incubator space and reducesmedium consumption

• Recommended fill volume 10 ml for OptiCellTM and 30 ml for OptiCellTM MAX

• Two resealing access ports provide closed growth environmentwith sterile fluid path, reducing risk of contamination

• Barcoded for easy tracking and automation handling



Transportation of Live Cells –OptiCellTM Mailer Kit• Safe and convenient - incubator to mailer to

incubator • Access to O2 and stable pH during shipping • No disturbing or dehydrating of cells needed • Less consumption of media

Hybridoma Antibody Production –OptiCellTM MAX Kit• Completely self-sealed sterile system • OptiCellTM lasts more than a month for

continuous antibody production • Allows harvesting of antibody 3 times a week

for higher antibody concentration and yield • Greatly reduces incubation space • Easy to use - no need to spin down cells

when collecting supernatants

Freezing & Thawing Cells• Effective and convenient short-term storage

of cells at –80 °C and long-term storage at–152 °C

• No trypsinisation is needed

• Quick recovery and growth after thawing

Biomagnetic Cell Separation –OptiMag Kit• Minimise cell manipulation during magnetic

cell separation – separate and continue cellculture in the same device. No centrifugationneeded

• Better proximity of magnet to cellpopulations for purer separation and higher yields

• Separation can be done with or without a laminar flow hood

• Compatible for use with Dynabead®

antibodies and MACS® antibodies fromMilteny

Cell Imaging & Staining• Cultures can be fixed and stained in situ, can

be stored for repeats • Membrane can be sectioned for small scale

staining• Membrane with fixed cells can be stored for

repeats

• Low background fluorescence; does notinterfere with fluorescent assays

• Eliminates need for inverted lenses

Transfection• No need to change the transfection reagents

or protocols• Easy to observe the transfection result under

a fluorescent microscope when markers areused

• Simplify the process of isolating single cells

Dynabead and MACS are trademarks of Miltenyi Biotech

*Application notes available

NUNC™ OptiCell™ Cell Culture System

APPLICATIONS*

Hybridoma Antibody Production in OptiCellTM Max


Troublesome effects caused by microbial contaminationMicrobial contamination, caused by bacteria, bacterial spores, viruses, mycetozoa, yeast or other

microorganisms, frequently presents a major risk in cell culture experiments. Since thiscontamination does not necessarily occur together with the overgrowth of the cultivated cell

type, it is often detected too late.

Changes in host cell morphology and even genetic changes such as chromosomalaberration and translocation, can, for instance, be caused by mycoplasma infection. Inextreme cases, a single germ can destroy the work of weeks or months of intricateresearch effort.

Why contamination control is essential…In view of the significant progress in the area of sensitive cell culture applications,such as tissue engineering or regenerative cell and tissue therapy, the requirementsfor CO2 incubators have changed.

Highest standards are thus applied to the perfection and reliability of the entireprocess chain, in which the CO2 incubator occupies a key position, since it must

replicate the natural in vivo conditions for optimal cell growth as accurately as possible.Sterile conditions must be guaranteed for in vitro cell cultures throughout the entire

cultivation period, since in addition to the risk of spreading contamination; the life-threatening danger of infecting patients is ever present.

What sterilisation really means…Let us briefly look at the concepts of sterilisation, decontamination and disinfection. Sterilisationstands for the complete elimination and/or absence of viable microorganisms; disinfection isunderstood as the elimination or inactivation of all pathogens present, which frequently onlyrepresent a partial quantity of all the present contaminants, however. The term decontamination,on the other hand, can be used in various ways such as for the removal of biological or chemicalor radioactive contamination, but it often does not allow any precise quantifiable conclusions tobe made with respect to its effectiveness.

The pharmacopeias basically specify autoclave sterilisation, hot air sterilisation and the use ofethylene oxide and sterile filtration as sterilisation methods. From these, only hot air sterilisationseems a feasible method suited to CO2 incubators.

For effective sterilisation, the various national pharmacopeias have agreed upon using a 6-logreduction of viable microorganisms, which is equivalent to one viable microorganism in a million,i.e. 1:1,000,000 units. This corresponds to a reduction of 99.9999% min. in the number of testorganisms that were initially used.


Standards compliant hot air sterilisation at 180 °C – a reliablemethod for contamination control in CO2 incubators

Figure 1. International standards of hot air sterilisationStandard Temperature (°C) Sterilisation period (min)US Pharmacopoeia 170 120European Pharmacopoeia 160 minimum 120 minimumAmerican Dental Association 160 120 ANSI/AAMI ST63-D ≥160 Not defined ANSI/AAMI ST50 160 120 DIN 58947 180 30 Pharmacopoeia Nordica 180 30 Hygiene directive 160 200 Robert Koch Institute 180 or 30 Japanese Pharmacopoeia 160 - 170 or 170 - 180 or 180 - 190 120 or 60 or 30British Pharmacopoeia 160 minimum 60 minimum

Figure 1. Applying hot air attemperatures >=160°C, as dry heat, atthe exposure times defined in thePharmacopoeia and validated byevidence of successful USP test germremoval would thus qualify as anapproved hot air sterilisation method.




Controlling contamination in cell and tissue culture incubatorsThe various manufacturers of CO2 incubators have developed some very different concepts forthe prevention and control of contamination; in this context, the requirement for sterility of a cellculture inside a CO2 incubator has posed significant technical challenges.

In selecting a suitable method, the following critical aspects must be taken into account:

1. That the inner chamber of the incubator is suitable for periodic spray/wipe disinfection, whichis the standard process to reduce the bio burden, i.e. the microbiological load of the CO2

incubator system. Easy to clean metal and glass surfaces (incubator interior, and the glassdoor which closes off the test space) that have no welding seams, and wherever possibleshould have no screw connections and/or elements which must be dismantled beforedisinfection (fan impellers, covers of air duct elements) to allow prompt cleaning and uniformwetting of all interior surfaces with disinfectant. Reducing the number of interior fittings suchas sliding rack systems, humidification systems, etc. to the absolute minimum technicalessentials in order to minimise potential contamination of inner surfaces right from outset.

2. Prevention of condensation which could serve as breeding ground for germs in the incubatorinterior.

3. Secure elimination of potential contamination by means of verifiable, effective sterilisationprocesses.

In addition, the cell culture system used should prevent the introduction of airborne germs, someof which are present even under clean room conditions. Cell culture bottles with a 0,2 µmbacterial filter were found to be suitable for this purpose.

The BINDER concept to minimise surface contamination andeffectively eliminate contamination

BINDER series CB and C 150 CO2 incubators are designed for easy spray/wipedisinfection and routine auto-sterilisation. This customised design facilitates applicationand does not require replacement of expensive parts, such as filters, UV lamps, etc. Itcontains the following components:

• Easy to clean seamless, deep-drawn inner chamber with 27% percent lesssurface for potential contamination, and an integrated shelf mounting system tominimise surface contamination

• Absence of condensation, even when working under conditions of highlysaturated air humidity, and mechanically polished stainless steel surfaces withoutwelding seams to prevent nesting of airborne germs

• Verifiable, effective automatic hot air sterilisation at 180 °C in compliancewith standards, which can be performed conveniently overnight and meetsinternational guideline requirements for hot air sterilisation.

Daniela Maurer, Dipl. Biochem., Scientific Product Management, BINDER GmbH, Tuttlingen

BINDER CO2 incubators

series CB and C150

>



The most trusted conical tubes are now easier to use! The BD Falcon™ 50 ml PolypropyleneConical Tube with Flip-Top Cap is the newest addition to the BD Falcon™ tube family.

The BD Falcon™ 50 ml Conical Tube with Flip-Top Cap is ideal for any research applicationsrequiring one-handed operation to open and close the cap. It also allows researchers to avoidhaving to remove the cap and either hold it, or place it on a surface away from the tube,before dispensing the contents of the pipette.

This novel design saves time and effort in applications requiring multiple aliquoting, storage,and pouring from the same tube, while maintaining the same superior quality andperformance of our standard screw-cap closure.


BD Falcon™ 50 ml Conical Tube with Flip-Top CapThe same tube you have always trusted with a NEW innovative cap design…

Shop!

>>new

THE FLIP-TOP CAP ADVANTAGE:

Easy-to-use – One-hand cap manipulation is virtually effortless with theunique hinge action of the flip-top cap. Easy-to-read blue graduations on thetube allow rapid volumetric assessment.

Splashguard – Innovative design of the cap allows opening and closing of the cap without risk of splatter.

Secure seal – Innovative click-bead ensures a tight seal every time.

Ambidextrous – Thumb grips on each side of the cap ensure a sterileopening technique.




BD Falcon™ 50 ml Conical Tube with Flip-Top Cap:Simply flip the cap open and pipette your sample.

Product description Qty/Pk Qty/Cs Cat. No.

NEW BD Falcon™ 50 ml polypropylene conical tubes with flip-top cap 22/bag 440 734-1437BD Falcon™ 50 ml polypropylene conical tubes 25/bag 500 734-0448

25/rack 500 734-0453BD Falcon™ 15 ml polypropylene conical tubes 50/bag 500 734-0451

50/rack 500 734-0452

KEY APPLICATIONS

• Cell pelleting and washing

• Gradients

• Sample and buffer storage

• DNA/RNA/Plasma preps

• Serial dilutions

• Sterile tissue collection

• Immunofluorescent assays

• Storage of chemicals andmany more.

For a demonstration, and for more information, please contact your localVWR sales office.

Our commitment to quality

Extensive biological, physical and chemical testing has been performed by BD Biosciences –Discovery Labware for qualifying the high-clarity, bioanalytical grade polypropylene for use inBD FalconTM tubes.

Non-toxic – Resins are selected via U.S. Pharmacopeia (USP) toxicity tests to ensure our tubes do not contain concentrations of bioreactive substancesthat could potentially interfere with biological experiments.

High strength – The BD FalconTM Conical Tube with Flip-Top Cap can becentrifuged up to 9400 x g.

Optical clarity – High-clarity polypropylene conical tubes provide easyviewing through ultraclear tube walls.

1. Flip open 2. Pipette 3. Mix 4. Ready


All immunoassays are characterised by abinding reaction between the target analyteand antibody. In theory, an antibody binds to asingle, specific antigen only. In reality,cross-reactivities, non-specific binding andmatrix effects negatively influence all assays.Interfering substances are present in more orless significant concentrations in specimen,interacting directly with the analytes, thecapture or the detector antibodies,respectively. Apparently well characterisedantibodies, with a high affinity to the targetanalytes, occasionally show surprising results.For example during immunological detectionon Western blotting membranes, unwantedbands are stained; on protein arrays oneobserves fluorescence signals for spots at thewrong positions from immobilised captureantibodies as well as a high backgroundsignal for blank samples. In ELISAs one gets ahigh background for the negative control orfalse negative signals during measurements.Known interfering factors, which are regularlydescribed in the technical literature, are e.g.heterophilic antibodies as well as HAMAs(humane anti mouse antibody) or rheumatismfactors, albumins, complement factors,lysozyme and others.

Interference byimmunoassay label

Usually, detector antibodies orstandard analytes are labelled withenzymes (e.g. alkaline phosphatase or(horseradish) peroxidase),fluorescence dyes, radioactive isotopesor DNA (immuno-PCR). Such labelsmay influence the affinity of antibody-antigen; reduce the solubility oflabelled proteins. Likewise, proteins orantibodies from serum samples canbind to fluorescence dyes and reduceor even switch off the fluorescence ofthe dye.

Interference by cross-reactions

Cross-reactions are the ability of the antibodyto bind to other structures than the target

analytes. Frequently, these structures show agreat similarity to the analytes. Examplesthereof are metabolites, chemical substanceswith a similar molecular structure, andfragments of degraded target proteins orproteins with a similar amino acid sequence.

Interference by unspecificbinding

Closely related to cross-reactivity is unspecificbinding. However, the causes on the molecularlevel differ. While the wrong binding partnersin the mechanism of cross-reactivity areknown, unspecific binding is caused bysubstances, which are in far excess of thetarget analyte (i.e. unspecific binding toalbumins or immuno globulins) or withsurfaces (i.e. surfaces of ELISA plates orWestern blotting membranes) or with spots ofimmobilised antibodies in protein arrays.

Matrix effects

Matrix effects are the sum of all interferingeffects of all components from the specimen,and influencing the measurement of a targetanalyte. Some matrix effects are derived from“anti-animal-antibodies” others fromheterophilic antibodies, from endogenousinterfering substances or just from viscosity,pH value or simply the salt concentration.

Interference by “anti-animal-antibodies”

Human anti-animal-antibodies (HAAA) can beof the IgG-, IgA-, IgM- or IgE-type. They arepart of the immune system's answer tocontacts with immuno globulins from animalorigin or contact of humans to pets and otheranimals. Human anti-mouse-antibodies(HAMA) are the most popular ones. Thereason for the presence of these antibodies inpatients are often therapeutic applications ofsuch antibodies (e.g. cancer therapy).Sequence similarities between antibodiesderived from different species may bedetected by HAMA-containing sera too. Someinterfering antibodies are not only directedagainst the Fc-fragment, but also against the


Optimisation of Immunoassays

Recognise and avoid interference in Immunoassays

Antibodies are useful tools to

specifically detect many substances.

Methods like Enzyme-linked

immunosorbent assays (ELISA),

Enzyme immunoassays (EIA),

Western blotting,

Radio immunoassays (RIA),

Protein-Arrays,

Immunohistochemistry

or the immuno-polymerase chain

reaction (Immuno-PCR) belong

to this category of the immuno

detection technology. Even if

they are highly specific, they all

have in common to be disturbed

by unwanted cross-reactivities.

By applying a new buffer

(CrossDown™ Buffer) instead of

the usually used buffers most

interfering effects can be avoided.


Fab-fragments of assay-antibodies. This canresult in a reduction or total hindrance ofcorrect binding, resulting in false-negativesignals. The binding of HAAAs to the Fc-fragment is called anti-isotypical, whilebinding to the highly variable Fab-fragment iscalled anti-idiotypical.

Interferences by heterophilicantibodies

Taber’s Medical Dictionary defines heterophilicantibodies as "antibodies, which bind otherantigens than the specific antigen".Heterophilic antibodies can be of the IgG-,IgA-, IgM- or IgE-type. Particularly the IgM-type plays a particular role in sera ofrheumatic patients. Rheumatism factors areIgM antibodies that bind to the Fc sections ofhumane antibodies and therefore bindspecies-independent to Fc sections ofantibodies used in the assay. Rheumatic serajoin capture with detector antibodies with theconsequence of false-positive signals. This is atthe same time the general interferingmechanism of the heterophilic antibodies. Theeffect of the rheumatic sera resembles theeffect of the HAAAs. The difference compared

to the HAAAs is the origin of the heterophilicantibodies: These are not build on contactwith animal immunoglobulins, but they aremulti-specific antibodies of the early immuneresponse or interfering antibodies withunknown immunological history or origin.

The Hook effect

The Hook effect leads to false-negativedeterminations that, however unlike the otherinterferences, do not arise through interactionswith interfering factors.A Hook effect canappear in assays, where the specimen is mixeddirectly with the assay antibodies and theanalyte is present in very high concentrations.In this case the high concentration of theanalyte, when it exceeds the concentration ofthe assay antibodies, saturates capture anddetector antibodies. Thus high concentrationssimulate far lower concentration in the assayand lead to a significant underestimation ofthe true concentration in the assay. The Hookeffect can be avoided in practice by usinghigher concentrations of the assay antibodiesor by dilution of the specimen.Alternatively, asystematic dilution of the test has to ensure,that the measured value is not subject to aHook effect.

Prevention of interferenceby CrossDown™ Buffer

The reasons for the described interference aresimilar. There are unwanted low to middleaffine interactions of the interfering factorswith the antibodies or the analytes.And thereis low or middle affine binding of labeledantibodies to other proteins or surfaces as well

as low to middle affine cross reactivities of theantibodies to structurally related substances.These interferences have something incommon that the newly developedCrossDown™ Buffer makes use of: Theinterference is weaker than the specificbinding of the real analytes. Of course thereare rare exceptions, since very high affinecross-reactivities can occur that achieve thesame quality as the real specific binding. Insuch cases one must speak about a specificrelationship and principally, one has anantibody that is aimed at two differentsubstances. Therefore such an antibody isuseless for specific assays. The CrossDown™Buffer was developed specifically to eliminateweak and middle affine binding, withoutnegatively affecting high affinity binding withhigh specificity in any way.It’s new that different interferences withdifferent molecular principles can beminimised with the same strategy.CrossDown™ Buffer is applicable for differentimmunoassays. CrossDown™ Buffer canprevent interfering effects by HAMAs andrheumatism factors, unspecific binding inimmunohistochemical applications as well asfalse-positive bands in Immuno-PCR.Takentogether, the time consuming and costly effortsfor optimisation strategies can be reduced andsimplified significantly whereas improvementsin reliability are attained simultaneously.



Figure 2. Western Blot without (left) and with (right) Blocking Solution I andCrossDown Buffer. Detection of Myostatin in mouse myoblasts with anti-GDF-8primary antibody and rabbit anti-goat IgG-HRP secondary antibody (asay andimage by Dipl.-Biol. Siewert, University of Ulm, Germany).

Figure 1. Reduction of unspecificbinding of a detector antibody to thesurface of a protein array. Improvementof signal-to-noise ratio from 3.4 (left) to17.3 (right) by the use of CrossDown™Buffer (data from N. Dankbar, Universityof Münster, Germany).

Figure 3. Prevention of false-positivebinding (control of specificity in row A1-12 and blank row H1-12) by the useof CrossDown™ Buffer in an ELISAagainst guinea pig IgG (by Dr. C. Specht,PARA Bioscience, Gronau, Germany).

with PBS

with CrossDownTM



The cooling and the freezing of biologicalproducts is a complex process during whichmany chemical and physiological changes canoccur. Biological products can be preserved inmany ways, but the storage at very lowtemperatures is the only method ofconservation that minimises these changes.Living cells have a critical temperature of -130 °C, beyond this threshold temperature the stability of the sample can’t beguaranteed. In order to guarantee stability ofthe sample living cells must be stored undertemperatures provided by using liquidnitrogen (-196 °C) and we then have a safetymargin of +60 °C from the critical point.

Storing in liquid nitrogen has obviousadvantages, however there are some risks tobe aware of when using this type of storagemethod:

Cross contamination Even if their number is limited, some cases ofcontaminations between different samplesstored immersed in liquid nitrogen have beenreported.

Leakage of liquid nitrogen into thevialsThere is always a possibility of explosion ofthe vials during their withdrawal from theliquid nitrogen even if this risk has beengreatly reduced thanks to the evolution of thequality of the plastics used.

SafetyDepending on their size, the racks filled withliquid nitrogen are sometimes heavy tohandle.

In order to solve the above risks linked to thestorage in liquid phase whilst stillmaintaining low storage temperatures it ispossible to store the biological samples in thegas phase of the liquid nitrogen. Laying a bedof liquid nitrogen into the bottom of thecontainer and using the gas phase to coolsamples, without the liquid contacting thesamples themselves, achieves this andminimises the problems associated withstoring in the liquid phase of liquid nitrogen.

Being concerned with the safety andpreservation quality of your biologicalproducts, Air Liquide DMC has developed anew and complete range of cryogeniccontainers to facilitate preservation in the gasform.

For the safety andpreservation quality ofyour biological products, Air Liquide DMC hasdeveloped a new completerange of cryogeniccontainers to facilitatepreservation in the gasform.

The ARPEGE Gas: new complete range of cryogeniccontainers




What’s new compared to the ARPEGEliquid containers?• The gas containers have a liquid nitrogen

background, completely regulated by anaccurate new capacitive gauge.

• A new range of racks has been developedtogether with a new liquid filling system

The advantages of the gaseous phaseProduct safety• The new level gauge regulates and

constantly maintains the amount of liquidnitrogen ensuring it is necessary andsufficient. Thus, products are kept in a gasatmosphere whose temperature is alwaysbelow -150 °C.

• Bags, cryotubes and straws used for thepreservation of cellular products aresensitive to thermal shocks which may be

accentuated when they change repeatedlyfrom the gas form to the liquid form, andback. In addition, penetration of a fewdrops of nitrogen into bags or cryotubescan trigger their explosion when this liquidis reconverted into gas.- In both these cases, using the gas form

prevents any deterioration of theproduct being kept.

- Contamination between products keptin one and the same container remainsmore difficult with a gas vector than aliquid vector.

User safety• When handling products, the storage

columns remain indisputably safer in thegas form, because it prevents anydischarge of nitrogen and hence any riskof burns.

The ARPEGE gas models 70, 110, 140 and 170

Vessel Total capacity Total capacity Straws Straws Straws CBS for 2 ml vials for 5 ml vials 0,25 ml 0,5 ml 0,5 ml

Arpege 70 1600 648 9520 3400 2380Arpege 110 3200 1296 19040 6800 4760Arpege 140 4200 1458 28560 10200 7140Arpege 170 5400 1944 42840 15300 10710

Main technical points for ARPEGE Gas phase containers

> How to transform your ARPEGE Liquid into ARPEGE GAS!• I have a liquid phase ARPEGE 70, 110, 140 or 170; can I transform it into

ARPEGE GAS? ➨➨ YES We’ve designed specific kits that include: level capacitive gauge, transfer tube &

electronic cabinet.

• Why should I replace my operating cabinet?➨➨ Special prices have been created specially for kits that include the

electronic cabinet!

• Can I use an ARPEGE Gas without the automatic refilling system? ➨➨ NO The Gas Phase preservation storage imposes for safety reasons an automatic refilling

system as the level of liquid represents ‘only’ about 10 cm of liquid.

• Do I lose a lot of storage capacity transforming an ARPEGE Liquid modelinto ARPEGE Gas? ➨➨ NO You lose only the lowest storage level.


PhotometryBacterial growth can be studied by continuousmeasurement of the optical density (OD) at awavelength area around 600-650 nm. Usingmicroplates in cultivation, the sample volumescan be decreased and the number of samplesincreased. Continuous kinetic measurementsare easy to perform with an instrument withan inbuilt incubator and a shaker. However,when the samples are grown on a microplatein elevated temperature, the condensationof water onto the microplate lid will causeproblems in continuous measurements asthe OD is measured through the lid. Withthe Varioskan Flash it is possible to carryout long-time measurements as theinstrument’s inbuilt incubator heats thelid to a slightly higher temperature thanthe other parts of the plate, thus avoiding

any condensation from occurring.The antimicrobial effect of a certaincompound can be studied by monitoringbacterial growth. In this assay Streptococcuspyogenes was cultivated with varyingconcentrations of antibiotics on a clear 96 well plate at 37 °C. The growth wasmonitored by OD measurements in 620 nmevery 10 minutes for 24 hours. The growthcurves of the bacteria are presented in Figure 1, showing that the Varioskan Flash issuitable for continuous OD measurements ofbacterial growth on a microplate with a lid.

Fluorescence IntensityThe PicoGreen dsDNA Quantitation Reagent(Invitrogen) is a well-known and sensitive

fluorescent nucleic acid stain for quantitationof double-stranded DNA (dsDNA).In a 96 well plate dozens of samples can beanalysed at a time. The dispenser of theinstrument can be used in dispensing thePicoGreen reagent to achieve the most

reliable and reproducible assay results. Figure2 presents the standard curve of thePicoGreen assay measured with theVarioskan Flash. In conclusion, the VarioskanFlash together with the PicoGreenQuantitation Kit is a practical tool fordetermination of DNA concentration.

Time-resolved FluorometryLanthanides are fluorochromes used in time-resolved fluorometry (TRF). An importantfeature of the lanthanide ions is their longfluorescent lifetime. When used as labels, thelanthanide ions are complexed with organicligands. The structure of the chelate orcryptate has an effect on the fluorescentproperties of the label. By measuring the TRF

Thermo Scientific Varioskan Flash is aspectral scanning microplate readerwith four detection technologies:photometry, fluorescence intensity,time-resolved fluorometry andluminometry. This paper presents theversatility of the instrument bydescribing some basic applications ofthe four different technologiesperformed with the Varioskan Flash.Photometric bacterial growthmeasurement, fluorometric PicoGreenDNA quantification, time-resolvedfluorescence spectra of lanthanidelabels and the DLR reporter geneassay with two luciferase enzymes are presented here.


Different measurement technologies of Thermo ScientificVarioskan® Flash offer versatility for selecting applications

Figure 1. Growth curves of Streptococcus pyogenesATCC 12351. Blue C6: control (no antibiotic), redC10: 0,0133 µg/ml erythromycin, green C9: 0,133µg/ml erythromycin. The x-axis iterations describemeasurements at 10 min intervals.

Figure 2. Standard curve of the PicoGreen assay

Figure 3. TRF excitation spectra (left) and emissionspectra (right) of the most commonly usedlanthanide labels: europium, samarium and terbium

Figure 4. DLR standard curves of Firefly and Renillaluciferase

1 3

2


spectra of the label, the optimal excitationand emission wavelengths can be chosen foreach assay. Fluorescence lifetime of the labelcan be determined by using the decaymeasurement function. The excitation andemission spectra of the most commonly usedlanthanide labels measured with theVarioskan Flash are presented in Figure 3.The spectral scanning and decay calculationsof the Varioskan Flash together with SkanItSoftware are advantageous features forassay development; all the important assayparameters can easily be optimised.

LuminometryLuminescent reporter gene systems are verycommonly used to study gene expressionand regulation. In dual reporter gene assaystwo different reporter enzymes aresimultaneously expressed and measured.One enzyme is referred to as theexperimental reporter that responds to theeffects of the experimental conditions.Another enzyme is normally used as the

internal control to eliminate experimentalvariability caused by, for example, cellviability, transfection or cell lysis efficiency,or even pipetting. The Dual-LuciferaseReporter (DLR) Assay System from PromegaCorp. is propably the most common dualreporter gene system. It uses two differentluciferase genes, Firefly and Renilla, from adistinct origin. The DLR standard curves ofthose two enzymes measured with theVarioskan Flash are shown in Figure 4. Theassay sensitivities with both luciferases arein attomole levels and both reactions givelinear responses over a range of more thansix orders of magnitude, making it possibleto measure extremely low gene expressionlevels from cell lysates.



Thermo Scientific clear MicrotiterTM plates for photometry• Clear universal and enhanced binding surfaces have excellent optical clarity • Enhanced binding surface has high binding properties and low binding variation

from well to well, plate to plate and lot to lot• Available in 96 well and 12 x 8 strip formats • Sterile 96 well plate +lid, individually wrapped• 384 plate for miniaturisation of assays

Description Qty Cat. No.

White Cliniplate 96, universal binding 50 735-0535White Cliniplate 96, sterile+ lid, individually wrapped 40 735-0624White breakable 12x8 strip plate, universal binding 50 735-0568

Black and white plates for fluorescence• Black Cliniplate 96 offers high signal to noise ratio and low cross talk in fluorometric assays • White Microlite+ surface has low background and low well-to-well cross talk -> improves

sensitivity of your PicoGreen or other lower signal fluorometric assays • White Microlite+ binding surface is available in 96 solid plate and 8x12 strip plate format • Available as medium and high binding surfaces • 384 plates for miniaturisation of assays

White MicrotiterTM plates for luminescence• White universal binding surface has lowest phosphorescence background and lowest

well-to-well cross talk in the market -> improves sensitivity of luminometric assays • White universal binding surface is available in 96 well solid plate and

12x8 strip plate format • Sterile 96 well plate + lid, individually wrapped


Cliniplate 96, universal binding 50 735-0531Cliniplate 96, enhanced binding 50 735-0546Cliniplate 96, sterile+ lid, individually wrapped 40 735-0572Breakable 12x8 strip plate, universal binding 50 735-0551Breakable 12x8 strip plate, enhanced binding 50 735-0540Immulon 1 B 384 plate, medium binding 50 735-0254


Black Cliniplate 96, universal binding 50 735-0533Black Microfluor 1 384 Plate, medium binding 50 735-0626Microlite 1+ 96 Plate, medium binding 50 735-0525Microlite 2+ 96 Plate, high binding 50 735-0526Microlite 1+, 8x12 strip plate, medium binding 100 735-0521Microlite 2+, 8x12 strip plate, high binding 100 735-0522White Microlite 1 384 Plate, medium binding 50 735-0258

4


The choice of the genomic DNA purification tool dependson the needs

A variety of different methods and technologies are available for the isolation of genomicDNA. Which method to choose depends on many factors: the required quantity and molecularweight of the DNA, the purity required for the downstream application, the processing timeand the expense.

The ArchivePure DNA Purification System is a highlyflexible and scalable salting out method

The ArchivePure DNA Purification System relies on biological or environmental specimens as asource of genomic, mitochondrial or viral DNA. Typical sample types include: whole blood,buffy coat, cultured cells, body fluids, animal or plant tissue, and microbes.

In the beginning of the preparation the crude lysate contains enzyme-inhibiting contaminants.These proteins and other contaminants are removed by precipitation using high concentrationsof salt. The precipitates are removed by centrifugation, and the DNA is recovered by alcoholprecipitation.

The high salt method is quick and reliable and can be routinely used for the extraction of highmolecular weight DNA. The archiveable DNA demonstrates unparalleled stability for manyyears without losing integrity and is perfectly suitable for all demanding downstreamapplications like Microarrays, Southern Blots, PCR or Real-time PCR. The reagents only systemis highly flexible because without using a column or beads, it’s scalable based on the startingsample volume.

The PerfectPure DNA Purification Kits simplistic designrelies upon silica-membrane columns

The PerfectPure DNA Purification Kits provide the components and procedures necessary for the purification of genomic DNA from whole blood, buffy coat or cultured cell samples.The resulting DNA is essentially free of RNA, protein, and other enzymatic inhibitors for use in demanding applications like Microarrays, Southern Blotting, PCR and Real-time PCR.

The simple and fast method is based on the selective adsorption of nucleic acids to a silica-membrane in the presence of a proprietary non-chaotropic reagent.The majority of silica-membrane kits rely upon chaotropic salts that can interfere with the


IItt’’ss ttiimmee ffoorr 55 PPRRIIMMEE - a wide choice of genomic DNApurification tools

Room Temperature: RIN mean 9,67273 +/- 0,417351 65°C/15 min: RIN mean 9,57500 +/- 0,476970

Figure 1. Agilent traces, andcorresponding RINs, arerepresenting PerfectPure purifiedRNA before and after 65 degreeheat for fifteen minutes

5 PRIME’s breadth ofgenomic DNA purificationtools:

1. Highly flexible and scalable salting-out method – ArchivePureDNA Purification System

2. Simple and reliable silica-membranemethod – PerfectPure DNAPurification Kits

3. Fast and unique filtration method –Ready PCR DNA Kits




downstream applications through salt carry-over. In addition, the use ofan optimised buffer for cell lysis ensures that common problems such asoverloading and clogging of the columns are avoided. From up to 10 mlblood a yield of up to 500 µg and from 25 million cultured cells up to200 µg high quality genomic DNA can expected in as little as 30 minutes.

Fast and unique filtration method of the Ready PCR DNA Kits provide ready-to-use PCR grade DNA

The Ready PCR DNA Purification System is a rapid system using a unique filtration method toprovide ready-to-use DNA for PCR screening.

The Ready PCR DNA Card and Disk Kits are for isolation of DNA from whole blood, bonemarrow, buccal cells, saliva and cultured cells on sample collection cards. The sample is appliedto a sample collection card and allowed to dry. The DNA is purified from the sample bypunching out a 3 mm disk, washing with the DNA Purification Solution and then dehydratingwith alcohol. During purification, the DNA remains bound to the disk and, following the dryingstep, the sample is ready for DNA amplification.

The Ready PCR DNA Column and Plate Kits employ a flexible solid phase system on a columnor 96-well plate with a unique trifunctional purification matrix and reagents that lyse cells andthen capture and release DNA. This method does not require pre-lysing of the sample orprecipitation of the DNA.

The cells contained in the sample lyse upon contact with the matrix. Once the cells are lysed,the cell lysate is captured by the matrix material that makes it possible to efficiently washcontaminants, leaving the DNA bound to the matrix. After removal of the contaminants, theDNA is released from the matrix using an elution solution and heat. The purified DNA iscompatible with PCR, but cannot be used for restriction digests or southern blot analysis.

Molecular biology

For more in-depth information on 5 PRIME’s genomic purification tools,please contact your local VWR sales office.

Genomic DNA from Product Size Cat No.

Whole blood, bone marrow, packed cells and buffy coat ArchivePure DNA Blood Kit 3 ml 733-1013Cells and fresh, frozen, fixed or paraffin-embedded tissue ArchivePure DNA Cell/Tissue Kit 100 mg 733-1017

ArchivePure DNA Tissue Kit 100 mg 733-1021Mouse tails ArchivePure DNA Mouse Tail Kit 100 mg 733-1024Buccal cells ArchivePure DNA Buccal Kit 10 Preps 733-1027Plant Tissue ArchivePure DNA Plant Kit 200 mg 733-1031Yeast & Gram-positive bacteria ArchivePure DNA Yeast & Gram + Starter Kit 733-1032PerfectPure DNA Purification KitsWhole blood, buffy coats PerfectPure DNA Blood Kit 50 Preps 733-1068Cultured cells PerfectPure Cultured Cell Kit 50 Preps 733-1071Ready PCR DNA Purification KitsWhole blood, buffy coat, plasma, body fluids and cultured cells Ready PCR DNA Column Kit – Purification on a filter-based column 10 Preps 733-1049

Ready PCR DNA Plate Kit – Purification on a filter-based 96 well plate 3 Preps 733-1056Ready PCR DNA Disk Kit – Purification on sample collection cards 10 Preps 733-1053

Buy one get one free offer in the

Shop!


Thermo Scientific has launched a novel solution to one of the major issues facing researchersin the field of DNA amplification. The repeated rounds of heating and cooling that occurduring the PCR process take their toll on the structural integrity of a PCR plate, causing themto loose their original shape. Once deformed, the PCR plates can become difficult to removefrom the instrument. Researchers often have to resort to drastic measures to recover theplate from their instrument, risking damage to their thermal block and loss of precioussamples.

Using two-shot moulded plates can eliminate warping, but as these are made from twodifferent plastics, by an expensive process that makes them more costly for scientists to buy.Thermo Scientific has come up with a more customer-friendly alternative. The new ultra-rigidABgene SuperPlate is made in a single process, from specially selected polypropylene to a newdesign specification. Consequently, the new ABgene SuperPlate offers customers greater valuethan all other PCR plate formats.

The ABgene SuperPlate is available in a range of colours – white, natural, blue, green, purple,red and yellow –that allows labs to use a separate colour for each PCR application or forindividual researchers to choose their own colour. Generally, PCR plates are embossed withgrid referencing to help researchers keep track of their samples. The grid referencing on theABgene SuperPlate 96 is printed black for easy visualisation and maximum convenience. Theunique combination of reduced warping, visualisation and high-performance makes theABgene Superplate the new gold standard for PCR applications, offering customers peace ofmind in their PCR experiments


Thermo Scientific ABgene® SuperPlate™ - Peace ofmind in a warped world!

Introducing the new Goldstandard in PCR plastics –delivering both higherperformance and greater ease-of-use.

✓✓ 4x more rigid than standardpolypropylene plates – ableto withstand automatedmanipulation withoutdeformation

✓✓ Enhanced thermal stability –improved frame design andspecially selected polymerminimises warping duringthermal cycling and afterheat sealing

✓✓ Raised black lettering – quickreferencing of wells allowstime saving and pipettingerrors

✓✓ Thin walled U-bottom wells –optimal heat transfer andmaximum sample recovery

✓✓ Optimised for QPCR –available in white to providethe highest level ofsensitivity and lowestvariance for QPCR

✓✓ Raised rim design – optimisedwell surface area improvesseal integrity for superior andconsistent sealing withoutcross-contamination orevaporation



For more information on these products contact your local VWR sales office,send an e-mail to [email protected] or visit our website www.vwr.comMolecular biology

FEATURES• Over 4x more rigid than standard polypropylene plates

• Ultra-rigid SBS footprint compatibility with automated systems

• Enhanced thermal stability to minimise warping during thermalcycling and heat sealing

• Black lettering for quick referencing of wells

• Thin walled U-bottom wells for optimal heat transfer and maximumsample recovery

• 0,2 ml maximum well volume (when used with adhesive and heatseals)

• Stackable design

• Sealable using Thermo-Mats™, Adhesive Films and Foils, Heat Sealsand Flat and Domed Cap Strips

• Skirt provides compatibility with automated systems

• Fits most thermal cyclers including the BioRad Opticon™ andOpticon™ 2 real-time instruments and the MegaBACE™ capillarysequencer

• Available in white for QPCR applications.

• Adapter plates for ABI PRISM® 3100 and 3700 sequencers available

Figure 1. Plate rigidity testingperformed using a tensometer todetermine stress required to deflectthe plate skirt by 2 mm and 5 mm.

0.0

500.0

1000.0

1500.0

2000.0

2500.0

3000.0

3500.0

4000.0

4500.0

5000.0

52

Deflection Distance (mm)

Stre

ss/ k

Pa

Standard Plate

ABgene® SuperPlate™

Description Colour Cat. No.

SuperPlate™ Skirted PCR Plate Colourless 732-1049 SuperPlate™ Skirted PCR Plate Blue 732-1050 SuperPlate™ Skirted PCR Plate Purple 732-1051 SuperPlate™ Skirted PCR Plate White 732-1052SuperPlate™ Skirted PCR Plate Yellow 732-1053


Results

1. The RNA electroporation systemimproved transfection efficiency andreduced or eliminated transfection relatedtoxicity. Over 90% transfection efficiencywas achieved for human PBLs and murineT cells stimulated with ConA or antigen-specific pepetide [1].

2. Optimisation experiments using differentelectroporation parameters for stimulatedhuman PBLs showed that the viability oftransfected T cells was in the 63-86%range 24 hours after electroporation witha transgene expression of 99% 3 daysafter electroporation. “Nucleofection, amodified electroporation method, has anumber of limitations. With thistechnology special buffers (supplied onlyby the manufacturer) are required to beused with a specific electroporationapparatus and preset programs limit theuser’s ability to develop alternativeelectroporation conditions”, YanbingZhao [1].

3. Cells stimulated from 2 to 18 daysshowed similar efficiencies indicating thatthe post stimulation time length doesn’tgreatly influence RNA electroporation [1].

ECM 830/ HT 200 System


High efficiency transfection of primary human andmouse T lymphocytes using RNA electroporation

Introduction

The difficulty associated with nonviral genetransfer methods in primary lymphocytes canresult in low gene transfer efficiency andhigh transfection related toxicity. Highefficiency gene transfection and lowtransfection related toxicity was achieved byelectroporation using in vitro transcribedmRNA. RNA electroporation may be used toengineer T cells with new biologicalfunctions, providing a new and powerful toolfor altering T cell biology where long termtransgene expression is not necessary ordesirable [1].

4. RNA electroporation shows advantagesover plasmid DNA based electroporation.The plasmid must enter the nucleusbefore DNA can be transcribed intomRNA following electroporation.Transcription efficiency is dependent onthe plasmid vector promoter, which canbe influenced by target cell type-specifictranscription factors, ultimatelydetermining transgene expressionefficiency. Transcription is bypassed byintroducing mRNA directly into thecytoplasm, and the target gene can beimmediately translated into protein. GFPexpression was detectable within 30minafter electroporation. A syntheticmolecule that mimics native mRNA’s isintroduced during RNA electroporation.Bacterial based plasmids contain signalreceptors that can lead to alterations in Tcell biology [1].

5. Optimised electroporation conditions didnot induce adverse effects on T cellviability and proliferation, or causeapoptosis [1].




Figure 1. Effects of electroporation on T cellgrowth and function. (A) 5 x 106 stimulatedPBLs were electroporated with increasingamounts of IVT GFP RNA as indicated. Sampleswere collected at different timespostelectroporation and the cell numbers werecounted and growth curves plotted. (B) PBLswere electroporated as in (A). Three days afterelectroporation, the numbers of annexin V- and7-AAD- positive cells (% of total cells) weredetermined. The results shown were plottedwithout gating (representative of twexperiments). (C) The melanoma tumor antigengp100-specific TCR-transduced PBL line (gp100-APB) or nontransduced PBLs as control (PBL)were electroporated with GFP (gp100-APB/GFPRNA) and compared to PBLs electroporatedwithout RNA (gp100-APB/NO EP). Seriallydiluted gp100 peptide-pulsed T2 cells werecocultured with the electroporated PBLs. IFN-gsecretion was detected by ELISA (representativeof two experiments).

Conclusion

RNA electroporation is a highly efficient toolto introduce genes into human and murineprimary T lymphocytes. This technology maybe used to engineer T cells with newbiological functions. The availability of a BTX96 well electroporation system makes thistechnology compatible with high throughputscreening protocols, and scalable geneticmodifications of T lymphocytes required forclinical applications. There are possiblecandidates that can be transferred into Tcells for the adoptive immunotherapy ofcancer and infectious diseases.

Reference1. Zhao,Y., Zheng, Z., Cohen, C., Gattinoni, L.,

Palmer, D., Restifo, N., Rosenburg, S., Morgan, R.,(2005).High Efficiency Transfection of Primary Humanand Mouse T Lymphocytes Using RNAElectroporation.Molecular Therapy 668.

Table 1. GFP expression of CD3+ cells and cell viability (by PIexclusion) were detected 24 hpostelectroporation. nd, not determined. Data are representative of two independent experiments.a Cryopreserved PBMCs (resting PBLs) were thawed, washed twice with OPTI-MEM, and electroporatedwith IVT GFP RNA at the settings indicated.b PBLs were stimulated with OTK3 for 3 days. Electroporation was conducted at the settings indicated.c Fresh murine splenocytes were electroporated with IVT GFP RNA at the settings indicated.

Settings (ms) Resting human PBLsa Stimulated human PNLsb Resting murine splenocytes

%GFP MFI Viability (%) %GFP MFI Viability (%) %GFP Viability (%)200 V/50 44 170 33 91 746 60 21 9200 V/20 69 176 62 93 536 78 18 41200 V/10 69 129 62 83 212 86 17 50300 V/10 95 286 57 nd nd nd 52 28300 V/5 nd nd nd nd nd nd 49 41360 V/5 88 214 47 nd nd nd 56 15360 V/2 nd nd nd 78 437 70 nd nd360 V/1 nd nd nd 90 153 81 29 52400 V/0,5 30 24 68 93 100 82 15 57500 V/2 nd nd nd 58 236 54 nd nd500 V/1 nd nd nd 90 227 63 nd nd500 V/0,5 nd nd nd 94 123 70 nd ndNo EP 0,5 8 73 0,6 4 62 0,5 72

Table. Optimisation of electroporation conditions for resting and stimulated T cells


µW/cm2

P-030-02Single UV Bulb

Workstation

200

P-036-02Single UV Bulb

Workstation

200

P-048-02 Single UV Bulb

Workstation

400

P-030-202Dual UV BulbWorkstation

400

P-036-202 Dual UV BulbWorkstation

400

P-048-202 Dual UV BulbWorkstation

800

The PCR Workstation™ is designed toprovide an optimal environment forperforming PCR amplification reactions. TheWorkstation interior can be irradiated prior touse, blocking replication of potentiallycontaminating DNA sequences by causingadjacent pyrimidines to undergo dimerisation(1, 2). The PCR Workstation™ is equippedwith either 1 (single) or 2 (dual) ultravioletlamp(s) that emit light with a 254 nmwavelength. When solubilised DNA isexposed to this radiation adjacent thyminebases will be induced to form cyclobutanepyrimidine dimers by the condensation oftwo ethylene groups at C-5 and C-6.Additionally, adjacent thymines can be linkedbetween the C-4 residue and the C-6 of itsneighbour. In either case, a “kink” isintroduced into the DNA. Therefore, by pre-exposing the PCR work area with UV radiation, all DNA present will be photo-damaged and will not be amplified by DNA polymerase (despite retaining theability to be primed). Consequently, thesephoto-damaged sequences will notcontaminate your PCR amplification product. (4).

The protected area within theWorkstation also limitsexposure of the experimentalset-up to the open labenvironment, decreasing thechances of cross or airbornecontamination.

The PCR Workstation™ isavailable in six differentmodels. The 3 sizesavailable are 61cm deep x61cm high, with widths of

76, 91, and 122 cm. Each size can be orderedwith either a single or dual UV light builtinto the ceiling. For added convenience, atwelve-hour timer controls the UV irradiationdosage, and can be set to a pre-determinedtime for decontamination. The PCRWorkstation™ can be placed on a labbench, or turned into a moveable work areaby ordering an accessory cart with lockingcasters.

Some labs may require the intensity of theDual UV Bulb PCR Workstation™ toadequately decontaminate their hood. TheDual Bulbs deliver twice the intensity of UVlight than that of the single, and will helpirradiate areas that might otherwise beinaccessible. The Dual UV Bulb format isrecommended when the researcher desiresto use the Workstation to decontaminateapparatus and reagents. Aerosols ofpreviously amplified DNA contaminants canoften be found on racks, pipettes, tubes, orreagent bottles. These potential sources ofcontamination need to be irradiated butwhen placed in a PCR workstation cancreate “shadow”areas where the UV dosagecan be insufficient for decontamination. Tohelp solve this problem, the two UV bulbsare mounted apart from each other in theceiling, maximising the contents of theWorkstation that will receive direct UVirradiation. The stainless steel ceiling andreflective white sides of the PCRWorkstation™ provide enough UV radiationbounce to get to all shadowed areas. TheDual Bulb format also shortens the amountof exposure time necessary by deliveringtwice the dosage of single bulb workstationsand may also be required to deliver the


PCR Workstation™ *

Protection against contamination in sensitive PCRamplification reactions

Table 1. Intensity of UV light for single and dual UV bulb PCR Workstation™


Shop!Receive a free replacementgermicidal light bulb and freereplacement fluorescent lightbulb with the purchase of any PCR Workstation™




sufficient UV dosage needed to preventreplication of certain types of contaminatingDNA. For example, inactivation of dry DNArequires more UV exposure than that of DNAin solution. To determine the best suitableUV irradiation dosage for the type of DNAbeing used please refer to Table 1 (above)and the information supplied in Ref. 3 tocalculate desired exposure times consideringthe type and source of contaminating DNA.

References1. Sakar, G., and Sommer, S, (1990) Nature 343

p. 27.2. Ou, C-Y, Moore, J.L. and Schochetman, G

Biotechniques (1991) 10:4 p. 442-445. 3. Fairfax, M.R., Metcalf, M.A., Cone, R.W., (1991).

PCR Methods and Applications. Cold SpringHarbor Laboratory Press, Cold Spring Harbor, New York. 1:142-143.

4. Lehninger, A.L., Nelson D.L., and Cox, M.M.,(1993) Principals of Biochemistry. Worth Publishers, New York, NY pages, 342, 816, 832, 837

Molecular biology

Description Cat. No.*

PCR Workstation™ 61 x 61 x 76 Single CBSCP-030-02-XL61 x 61 x 76 Dual CBSCP-030-202-XL61 x 61 x 91 Single CBSCP-036-02-XL61 x 61 x 91 Dual CBSCP-036-202-XL61 x 61 x 122 Single CBSCP-048-02-XL61 x 61 x 122 Dual CBSCP-048-202-XL

AccessoriesWorkstation table, 71 cm(h) for PCR Workstation™ P-030 730-0794Workstation table 94,4 cm(h) for PCR Workstation™ P-030 730-0795Workstation table, 71 cm (h) for PCR Workstation™ P-036 730-0796Workstation table 94,4 cm (h) for PCR Workstation™ P-036 730-0797Workstation table, 71 cm (h) for PCR Workstation™ P-048 730-0798Workstation table 94,4 cm (h) for PCR Workstation™ P-048 730-0799Table casters, set of 4 locking swivel casters 730-1815

*PLEASE NOTE: Electrical Outlet Specification must be added to end of the catalogue number. Pleaseadd the letter listed below that corresponds to the country desired: A: Norway, Sweden, Finland,Germany, Portugal, Austria, Netherlands, Luxembourg, D: United Kingdom, Ireland E: Denmark. F: France, Belgium, Spain. I: Italy. L: Switzerland

PCR Workstation™ is a trademark of C.B.S. Scientific Company

>

Each PCR Workstation™ is equipped with thefollowing:• Acid-resistant black formica work surface

• Tempered safety glass fascia

• Duplex electrical outlet mounted in ceiling

• Two fluorescent lamps mounted in ceiling assuringexcellent work space visibility

• A stainless steel ceiling is used for greater UV reflection,resulting in a 10% increase in UV dose in PCR Workstationswith a single UV bulb and a 20% increase with a dual UVbulb

• Single or dual 254 nm UV Germicidal lamp(s) with 12 hourcountdown timer with time-hold position

• Single or double doors can be closed to preventcontamination, and can be conveniently stowed awayduring experimental procedures by sliding into storagecompartment in the base of the PCR Workstation™

• Hinged glass fascia allows easy access to interiorworkspace for cleaning and placement of large instruments



Fast, reliable and high-quality large-scale plasmidisolation using E.Z.N.A.® Fastfilter® Plasmid System

TY ZHU, Guo Qi

We performed a comparative evaluation ofthe Fastfilter® Midi & Maxi Plasmid with twoother competitor’s comparable kits. TheFastfilter® Midi & Maxi Plasmid kits showedgreater yield and significant time saving overthe competitor’s kits.

We compared the yield, purity, process timeand the quality of the plasmid DNA preparedwith Fastfilter® Plasmid DNA kits with that ofother two kits from competitors.

Method Overview

The Fastfilter® Plasmid system uses three-step plasmid purification process(Figure 1) consisting of a modified alkalinelysis procedure coupled with lysateclarification using Fastfilter® syringe unit;a fast plasmid binding on spin column; andfinally the purified plasmid is eluted with lowsalt buffer (water or Elution Buffer).

Yield and Purity

The superior capacity of the Midi & Maxi spincolumn and Fastfilter® Lysate ClearanceSyringe used in the Fastfilter® Plasmid systemprovide overall better yield of plasmid DNAthan comparable products from twocompetitors with both low copy and high copynumber plasmid (Table 1, 2 and Figure 2).

Plasmid DNA samples isolated withE.Z.N.A.® Fastfilter® Plasmid System andcompetitors are all showed good results onthe UV spectrophotometric reading. Allpurified plasmid DNA samples exhibittraditional acceptable range on A260/A280ratio between 1,8–2,0, an A260/A230 ratioof 1,8-2,2. Table 1 also shows the averageyield and purity of plasmid DNA samplespurified from 50ml of an overnight culture ofDH5a cells. Table 2 shows the average yieldand purity of plasmid DNA from 200 ml of anovernight culture of DH5a cells. All the data

Table 1. Yield and purity from Midi kits (50 ml culture)Kits Vector Yield (µg) A260/280 A260/230

Fastfilter® Plasmid Midi Kit Psp65 160 1,82 1,92PEGF 259 1,86 1,96PET-32a 200 1,86 2,12Pgem-T 140 1,83 2,25

Supplier Q pEGF 200 1,85 2,02Supplier G pEGF 180 1,86 1,95

Table 2. Yield and purity from Maxi kits (200 ml culture)Kits Vector Yield (µg) A260/280 A260/230

Fastfilter® Plasmid Midi Kit pSP65 700 1,83 1,99PEFG 1000 1,80 1,96PET-32a 800 1,89 2,02Pgem-T 650 1,85 2,22

Supplier Q pEGF 950 1,84 2,02Supplier G pEGF 940 1,82 1,95

The Fastfilter® Plasmid Midi &Maxi kit are designed for thepurification of large-scale plasmidDNA with high quality. The newlydeveloped filter syringe and highcapacity spin column included inthe kits allow for the purificationof large yield of plasmid DNA inless than 30 minutes. With highcopy number plasmid, the highcapacity of the spin column canpurify up to 200 µg (Midiprep)from 50 ml overnight culture andup to 1 mg (Maxiprep) from 200ml overnight culture.

KEY FEATURES

✓✓ High Yield: Up to 200 µg(Midi) and 1 mg (Maxi) withexcellent reproducibility.

✓✓ High Speed: Large scaleplasmid isolation in less than30 minutes.

✓✓ High Purity: Purified plasmidsuitable for most downstreamapplications

✓✓ Convenience: Eliminate the time consumingcentrifugation with filtersyringe unit

Figure1. Flow diagramof plasmid DNA isolationand purification usingthe E.Z.N.A.® FastfilterPlasmid Systems.




(showed in Table 1 and Table 2) demonstratethat the presence of protein, salts, and RNAcontaminants were negligible. Agarose gelelectrophoresis is also used to confirm thequality of purified plasmid DNA (Figure 2).Figure 2 demonstrates that the plasmid DNAisolated with E.Z.N.A Fastfilter® Plamsid kitsare free of contamination of RNA andgenomic DNA. It also demonstrates that highpercentage of supercoiled monometricplasmid were isolated from all the tested kits.

Process Time

By eliminating the centrifugation step on thelysate clearance, coupled with fast processspin column format, the whole process ofFastfilter® Plasmid Midi/Maxi protocol can befinished in less than 30 minutes (vacuumprotocol). The process time for the Midi and

maxi kits from Supplier-Q and Supplier-G arenormally over 120 minutes (Figure4).

Downstream Applications

The plasmid DNA isolated with all three kitscan be completely digested with selectedrestriction enzyme (Figure 5). The plasmidDNA was also suitable for PCR andautomated sequencing applications withequivalent read length (based on Phred20quality scores) for all tested kits (Figure 6).

Summary

The Fastfilter® Plasmid Midi & Maxi kit cansignificantly reduce the time for thepreparation of large-scale of plasmid DNAwith equivalent DNA quality compared totwo major suppliers.

Molecular biology

Figure 5. Restriction enzyme digestion ofhigh copy number plasmids. The plasmidvectors pGEM-T easy, pEGF, pSP65 isolatedfrom E. coli DH5alpha® using the E.Z.N.A.®

Fastfilter Plasmid Systems, were digested with10 units of EcoR I or 10 units of BamH I andHind III for 1 hour at 37 °C. The digestedsamples were resolved on a 1% agarose gel,stained with ethidium bromide. Lane p1,uncut pGEM-T by Midi Kit; Lane e1, EcoR I cutpGEM-T by midi kit. Lane p2, uncut pGEM-Tby Maxi Kit; Lane e2, EcoR I cut pGEM-T bymaxi kit. Lane p3, uncut pEGF by Midi Kit;Lane e3, BamH I cut pEGF by midi kit. Lanep4, uncut pEGF-T by Maxi Kit; Lane e4, BamH Icut pEGF by maxi kit. Lane p5, uncut pSP65by Midi Kit; Lane e5, Hind III cut pSP65 bymidi kit. Lane p6, uncut pEGF-T by Maxi Kit;Lane e6, Hind III cut pSP65 by maxi kit.

Preps Cat.No.

E.Z.N.A.® Fastfilter Plasmid Midi Kit2 preps OMEGD6905-005 preps OMEGD6905-0125 preps OMEGD6905-03100 preps OMEGD6905-04

E.Z.N.A.® Fastfilter Plasmid Maxi Kit2 preps OMEGD6924-005 preps OMEGD6924-0125 preps OMEGD6924-03100 preps OMEGD6924-04

Preps Cat.No.

E.Z.N.A.® Fastfilter Endo-Free Plasmid Midi Kit2 preps OMEGD6915-005 preps OMEGD6915-0125 preps OMEGD6915-03100 preps OMEGD6915-04

E.Z.N.A.® Fastfilter Endo-Free Plasmid Maxi Kit2 preps OMEGD6926-006 preps OMEGD6926-0125 preps OMEGD6926-03100 preps OMEGD6926-04

Figure 2. 1,0% agarose gel analysis ofplasmid isolation by E.Z.N.A.® Fastfilter®

Plasmid System from DH5a (pET-32a).

Figure 3. Comparison of plasmid DNA yieldsusing different Midiprep and Maxiprepplasmid isolation systems. Plasmid DNA wasisolated following each manufacturer’srecommended protocol. All samples weredone in duplicate.

Figure 4. Comparison of time required permidiprep and maxipreps using differentsystems. Each system protocol was performedaccording to the manufacturer’s instructionsusing 50 ml (Midiprep) and 200 ml (Maxiprep)of an overnight culture of DH5a bacteriatransformed with a high-copy plasmid (pGEM®-T easy). For the E.Z.N.A.® Fastfilter PlasmidSystem, the vacuum protocol was used.

Figure 6.


GenoSmartFeaturing the new GenoSmart gel documentation system from VWR, analysis anddocumentation of your DNA and protein gels becomes very easy. By simply transferring thedata on to a USB stick you can work with your images on a separate PC without blocking thegel documentation system for other users.

• Real-time images from 8-bit CCD camera, (768 x 582 pixel resolution)• Self-contained, microprocessor controlled darkroom• Integrated colour LCD screen for image viewing• White Epi-light• Instant on-screen saturation detection• Rapid photographic print production• Transilluminator (optional)• White light converter (optional)

The GenoSmart darkroom allows you to use many bench top UV transilluminators – even oneyou might currently have in your laboratory. The system is compatible with most commonlyused electrophoresis gels stains and by using a white or blue light converter you can alsocapture images from other applications such as petri dishes, cells in flasks, autoradiographs,membranes, spot and slot blots of DNA, RNA or protein, TLC plates and more.


The VWR Collection introduces a new,complete rangeof Gel Documentation systemsThis broad, complete range provides a solution to every need

Gel documentation system GenoSmart Cat. No.

Digital video system with integral LCD monitor.Fits any benchtop transilluminator (not supplied). 730-3000

Transilluminators Size (cm) Wavelength (nm) Cat. No.

GenoView 20 X 20 254 730-301720 X 30 254 730-301825 X 30 254 730-301920 X 20 254 /365 730-302020 X 30 254 /365 730-302125 X 30 254 /365 730-302220 X 20 254 / 312 (302) 730-302320 X 30 254 / 312 (302) 730-302425 X 30 254 / 312 (302) 730-3025

Special introductory priceson the GenoSmart model in

Shop!

GenoPlex and GenoPlex ChemiThe GENOPLEX systems are available in a range of standard configurations for all fluorescenceand chemiluminescence applications. You can choose from models featuring the very latesthigh-resolution CCD cameras for use in general gel documentation or the more advancedspecification units for chemiluminescence work. Motor driven lenses are standard to bothsystems and you get the choice of selecting either manual or computer driven filter selectorsand a range of lighting options for both reflected and transmitted light applications. Thecamera/lenses, lighting and darkroom functions are computer controlled.

For more information contact your local VWR sales office.




Contact your local VWR sales officefor advice and a demonstration.

New range!

Image Capture couldn’t beeasier with the Genomini The GenoMini offers reproducible, highresolution digital images for geldocumentation. GenoMini is ideal if youhave a limited budget because you canpurchase the basic system and then add aUV transilluminator, white light table orconversion screen, safety hood, minidarkroom and printer, as well GenoSoftanalysis software.

• Remove camera lens cover ring• Charge camera battery• Assemble the camera lens adapter• Install camera software & set• Camera • Install camera software following the instructions in the manual, the camera should be set

to take black & white images • Attach camera to bracket • Assemble hood

Attach the camera bracket with camera attached to the top of the hood with the 2 screwsand washers provided. Align the camera with the hole of the hood (making sure the lightsealing foam ring is centrally positioned in the hole). The system is now ready for use; placethe camera/hood assembly on the transilluminator over the gel you want to image.

• To capture an imageTo capture an image connect the camera to the USB port of the PC. Select the ‘P’ option onthe camera main selector if not already set. Turn on the camera. This will launch thesoftware. Select remote shooting, press start and select the directory you wish to store theimages in. If your bands are faint then you may need to change the exposure compensationup in order to view them better. Select shooting settings to do this. Once happy with thelive image on screen click on the release button.

• Images are saved as JPEG files. If you wish to save as a BMP or TIFF file use the file exportoption. To import a file into GenoSoft for analysis start GenoSoft and select File/New(Import). Analysis is covered on a separate quick guide.

FEATURES

• Cost-effective, quality digitalimaging

• High resolution camera

• Flexibility to add hardware andsoftware

• Innovative mini-darkroom option

• Programmable and reproduciblecapture settings

• Real-time imaging using your PCor Mac


GenoMini 730-3002



As with all Axygen products, our PCR linesare precision moulded from 99,9% pure,virgin polypropylene and are certified RNase,DNase and endotoxin free for highestsample integrity. They are free fromlubricants, dyes, and heavy metals or fillers.Axygen recommends Maxymum RecoveryPCR products for applications requiringultimate performance.

Axygen tests and certifies each and everymanufacturing lot of products in it’s state-of-the-art onsite microbiology labwhich is fully equipped with the latest

instruments and technology. Experience anddedication to quality ensure that Axygenapplies the most stringent testing standardsto certify products free of contaminates suchas RNase, DNase, endotoxins (pyrogens),heavy metals and extractables. Axygenmaintains a validated sterilisation program,and certifies sterile products to have an SALof 10-6.

PCR plates, mats and seals are packed inresealable plastic bags with individual boxesto keep them free from contamination andfor easy storage.

Plates and mats for Real-Time PCR and sequencing

Axygen's extensive line of PCR products (Polymerase Chain Reaction) are

specifically designed for use within PCR thermal transfer and real time PCR

applications. The product line includes thin-wall tubes, 8-strip tubes/caps,

and a series of multiwell plates designed to fit virtually all thermal cyclers

available. A wide variety of plate sizes, configurations and colours are

offered to help organise multi-step PCR processes. Bar coding is available

for all skirted plates.




• All products are designedspecifically for Real-Time PCR

• Engineered and tested on the mostpopular thermal cyclers

• Plate wells have ultra-thin,consistent wall thickness

• All lots undergo the most rigorousQA/QC procedures in the industry

• Made from ultra-smooth diamondpolished moulds

• Assures optimal performance inevery well, every reaction

• Provides a secure fit regardless ofthe brand of equipment being used

• Gives precise thermal transferalong the entire surface of eachwell

• Ensures consistent, reliablecontamination-free performance

• Reduces sample “hang-up”reducing waste and improvingyields

Description – type of plates – working volume Axygen Ref Pack Cat. No.96-well plate and mat for 0,2 ml thermal cycler blocks

No skirt – 300 µl PCR-96-C 10 732-0661No skirt, elevated wells - 300 µl PCR-96-MB-C 10 732-0662No skirt – qPCR compatible – 300 µl PCR-96LP-FLT-C 25 732-1024No skirt, low profile, qPCR compatible, clear – 220 µl PCR-96-FLT-C 25 732-1021No skirt, low profile, qPCR compatible, black – 220 µl PCR-96-LPFLTBK 25 732-1023No skirt, low profile, qPCR compatible, white – 220 µl PCR-96-LP-FLTW 25 732-1022Segmented – 280 µl PCR-96-SG-C 10 732-0663Elevated skirt – 300 µl PCR-96-AB-C 10 732-0665Half skirt, low profile, qPCR compatible, clear – 220 µl PCR-96-LP-AB-C 25 732-1015Half skirt, low profile, qPCR compatible, black – 220 µl PCR-96-LP-ABBK 25 732-1016Half skirt, low profile, qPCR compatible, white – 220 µl PCR-96-LP-AB-W 25 732-1017Half skirt, qPCR compatible – 300 µl PCR-96M2-HSC 10 732-0803Full skirt – 200 µl PCR-96-FS-C 10 732-0664Compression Mat, 96 well, for Real-Time PCR CM-96-RT 10 732-1020Sealing mat for 96-well PCR microplates AM-96-PCR-RD 10 732-066896-well silicone compression mat CM-96-RD 10 732-067196-well silicone expansion mat CM-96-EXP 10 732-0672UltraClear film, pressure sensitive UC-500 100 732-0657


Shop!


Hollow Fibre Filtration

MediaKap® filters are designed to dramatically reduce the time required for sterilising andclarifying culture media and other hard to filter aqueous solutions. The flow rate of theMediaKap® filters is faster than you have experienced before. All MediaKap® units aremanufactured with the DynaFiber®, a 0,2 µm, micro porous, naturally hydrophilic, hollow fibremembrane. The hollow fibre membrane works in exactly the same fashion as the flat sheetmembrane you are familiar with. Filtration takes place through the wall of the fibre and thefiltrate is passed through the lumen (inside diameter) of the fibre (Figure 1).

Unique autovent feature

The use of the DynaFiber® results in high flow rates, low extractables and excellentbiocompatibility. Each unit contains a unique autovent feature which prevents airlocking andallows for operation of the filter in any position. The fibre bundle in each MediaKap® contains one hydrophobic fibre which acts as a downstream vent. When air gets into the line, it passesharmless downstream instead of filling the housing and blocking flow. Because its automatic,you do not have to remember to manually vent the filter as is necessary in flat disk filters.Also the vent fibre is 0,2 µm so the air passing downstream is sterile and will not contaminatethe downstream filtrate.

Designed as a pressure filter, MediaKap® may be used with a peristaltic pump, pressure vessel orrepeating syringe. All filter sizes are available with a filling bell to prevent cross-draftcontamination while filling under a hood. Manufactured to the highest standards, MediaKap® isnon-pyrogenic, 100% integrity tested and passes USP XXI Class VI toxicity testing.

Filtration of media with or without Serum

There are two types of filters:MediaKap® and MediaKap® PLUS.MediaKap® Filters are manufacturedwith the standard DynaFiber® hollowfibre and MediaKap® PLUS Filterscontain the high performance fibre(DynaFiber® HP) which hassignificantly greater throughout and is intended for media with serum andpartially soluble nutrients (i.e. soybroth, BSA, yeast extract). Typically,MediaKap®, type 5, will sterilise

5 litres of serum free media in less than 15 minutes and MediaKap® PLUS, type 5, will sterilise 5 litres of DMEM with 10% Fetal Bovine Serum in 20 minutes or less (Figure 2).

The hollow fibre takes up less space so the hollow fibre membranes can pack more surface areainto a given module than the flat sheet manufactures. As an example, one of the more popularsmall volume media filter on the market has a flat sheet membrane wrapped into a cylinder andcontains 10 cm2 of surface area. The MediaKap® 10 has a surface area of 100 cm2. Because thehollow fibre does not require a downstream support structure, hollow fibre membranes havehigher rates than flat sheet membranes.


MediaKap® hollow fibre media filters for sterile filtration of media with and without serum

PRODUCT SPECIFICATIONS:

2 Filter types MediaKap® &MediaKap® PLUS

5 volume sizes 2, 5, 10, 25 and 50 l

Pore rating 0,2 µm

Membrane SA see the orderinginformation

In-/outlet see the ordering connections information

Packaging Gamma irradiated

COMPONENTS

HF membrane Mixed cellulose ester

HF vent Polypropylene

Housing Clear polysulphone

End caps Clear & pigmentedpolysulphone

Potting material Polyurethane

Filling bell PVC(if present)

Figure 2

Figure 1



For more information on these products contact your local VWR sales office,send an e-mail to [email protected] or visit our website www.vwr.comSample preparation

PERFORMANCE SPECIFICATIONSWater Flow DMEM DMEM Surface

@ 0,7 Bar (ml/min) with serum (l) serum free (l) area (cm2)

MediaKap®-2 400 N.A. 0,2 to 2 35MediaKap®-2 Plus 400 0,2 to 2 5 35MediaKap®-5 750 N.A 2 to 5 70MediaKap®-5 Plus 750 2 to 5 10 70MediaKap®-10 1.000 N.A 5 to 10 100MediaKap®-10 Plus 1.000 5 to 10 20 100MediaKap®-25 1.400 N.A 10 to 25 185MediaKap®-25 Plus 1.400 10 to 25 50 185MediaKap®-50 2.000 N.A 25 to 50 440MediaKap®-50 Plus 2.000 25 to 50 100 440

CONNECTORS:Inlet Outlet

MediaKap®-2 / - Plus Female Luer Lock ¼ HB MediaKap®-5 / - Plus Female Luer Lock ¼ HB MediaKap®-10 / - Plus ¼ Hose Barb ¼ Hose Barb MediaKap®-25 / - Plus Variable Hose Barb Variable Hose BarbMediaKap®-50 / - Plus Variable Hose Barb Variable Hose Barb

FEATURES BENEFITS• 0,2 µm DynaFiber® or DynaFiber® HP • Biocompatible

(for MediaKap® PLUS) hollow fibre, • Fast processing timesmixed ester membrane • Free passage of soluble protein

• Small size • Less bulky than filters of equivalent filtration area• Automatic vent • Prevents air locking• Radiation sterilised • No ETO residuals• With or without filling bell • Maintains sterility• Hydrophilic fibres • No surfactants used• Tested and approved • 100% integrity tested

• All components meet USP XXI class VI toxicity standards• Non-pyrogenic by LAL

Description Filling Bell Qty/Pk Cat. No.

MediaKap®

MediaKap-2 Yes 12 734-0869MediaKap-2 No 18 734-0868MediaKap-5 Yes 12 734-0871MediaKap-5 No 18 734-0870MediaKap-10 Yes 12 734-0873MediaKap-10 No 18 734-0872MediaKap-25 Yes 6 734-0874MediaKap-50 Yes 3 734-0875

MediaKap® PLUSMediaKap-2 PLUS Yes 12 734-3570MediaKap-2 PLUS No 18 734-1324MediaKap-5 PLUS Yes 12 734-3576MediaKap-5 PLUS No 18 734-3571MediaKap-10 PLUS Yes 12 734-0877MediaKap-25 PLUS Yes 6 734-0878MediaKap-50 PLUS Yes 3 734-0879


The Transferpette® S -8 and Transferpette® S -12 are the newest additions to thesuccessful Transferpette® S range. Developed to fulfil the requirements of thestrictest life science applications, the instruments combine the most importantfeatures to become an exceptional standard in the pipetting field.

Filling microplates with multichannel pipettes is one of the common tasks in many modern lifescience laboratories. People working in these labs are faced with an increasing demand tocalibrate their multichannel instruments on a regular basis. To help doing this both theTransferpette® S -8 and Transferpette® S-12 offer the unique Easy Calibration technique.Unlike other pipettes the new Transferpette® S offers the possibility to calibrate the instrumentwithin seconds without any tools and without additional trial-and-error test runs forconfirmation.

This makes the Transferpette® S ideal for all laboratories working according to ISO 9001,GLP/GMP and ISO 17025.

In addition the new Transferpette® S-8/-12 manifold offers the possibility to remove andreplace shaft and seals in the lab for cleaning and repair. This patented procedure combinedwith the Easy Calibration technique eliminates expensive and long outages providing longservice life and low operating costs!


Transferpette® SNow also available as multichannel pipettes!

Solutions for cience



For more information on these products contact your local VWR sales office,send an e-mail to [email protected] or visit our website www.vwr.comLiquid handling


free single channel pipette with every multichannel purchased!

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No. of channels Volume (µl) Accuracy ≤≤ ± % Coefficient of variation ≤≤ % Cat. No.

8 0,5 - 10 1,6 1,0 613-37008 5 - 50 0,8 0,4 613-37018 10 - 100 0,8 0,3 613-37028 20 - 200 0,8 0,3 613-37038 30 - 300 0,6 0,3 613-370412 0,5 - 10 1,6 1,0 613-370512 5 - 50 0,8 0,4 613-370612 10 - 100 0,8 0,3 613-370712 20 - 200 0,8 0,3 613-370812 30 - 300 0,6 0,3 613-3709

Transferpette® S -8/-12

Items supplied: Transferpette® S -8 or Transferpette® S -12, conformity certified,performance certificate, 1 filled Tip-Box N, 1 Tip-Rack refill unit, 1 shelf/rack mount,1 reagent-reservoir, 1 set of sealing rings made of FKM.

More highlights!✓✓ Central pipetting button and separate tip ejector

✓✓ Ergonomic finger rest that adapts to your hand for arelaxed grip

✓✓ One-handed operation – setting and locking the volume,pipetting and ejecting – all without change of handposition!

✓✓ Completely autoclavable at 121°C (20 min.)

✓✓ Easy-to-read 4-digit volume display for highest precision,always clearly visible

✓✓ Compatible with most common tips

✓✓ CE-IVD-compliant


Molecular BioProducts brand offers brand market leading pipette tips.

The round and flat Gel tips, as well as the new duck billed Gel tips with

patented technology, give the Gel tip users simplicity of use and relieve

the frustration of ruined experiments. Molecular BioProducts brand Gel

tips are made with a long, soft fine point that will not endanger your

samples as compared to the blunt and hard point of standard pipette

tips. The new Gel tip line comes available in six styles as well as six outer

diameters to cater for every lab’s specific gel loading needs.

Work at temperature equilibrium

Allow liquids and equipment to equilibrate to ambient temperature.

The volume of sample delivered by air displacement pipette varies with

air pressure, relative humidity, and vapour pressure of the liquid, all of

which are temperature dependent. Working at a single, constant

temperature minimises the variation.


ART® and HLT™ Gel Tips from Molecular BioProducts

Universal Gel tipsApplication Description Volume (µl) Suitable for OD (mm) ID (mm) Sterile Racked Pack Cat. No.

loading gel thickness (mm)

DNA sequencing gels HLT Micro-Flat Gel 10 10 Up to 0,2 0,17 N/A X 200 tips per tray/ 732-22571 tray per pack

Agarose, ART Gel 20P 20 Up to 0,6 0,61 0,31 X X 96 tips per tray/ 732-22292-D & SDS PAGE, 10 trays per pack/ proteins 5 pack per case

ART Gel 100 100 Up to 0,6 0,61 0,31 X X 732-2226

HLT Round Gel 200 200 Up to 0,6 0,61 0,31 X 204 tips per tray/ 732-10545 trays per pack

HLT Round Gel 200 200 0,6 and over 0,57 0,38 X 200 tips per tray/ 732-225583 mm in length 5 µl, 1 tray per packgraduated

Ideal for polyacrylamide HLT Flat Gel 200, 200 Up to 0,4 0.37 N/A 732-2256& DNA sequencing gels 83 mm in length 5 µl,

graduated

732-2226 732-2229

Available in 10, 20,100 and 200 µl sizeswith the ART self-sealing barrier for 20 µl and 100 µl volumes.




Eppendorf style gel tipsApplication Description Volume (µl) Suitable for OD (mm) ID (mm) Sterile Racked Pack Cat. No.

loading gel thickness (mm)

Agarose, 2-D HLT Round Gel 10 10 0,66 and over 0,57 0,38 x 200 tips per tray/ 732-2337& SDS PAGE, proteins 1 tray per pack

Capillary systems HLT Ultra Micro Gel 10 10 0,4 and over 0,25 0,13 x x 204 tips per tray/ 732-10485 trays per pack

Agarose, 2-D & HLT Gel 10 Round Gel 10 0,66 and over 0,58 0,31 x x 204 tips per tray/ 732-1055SDS PAGE, proteins Loading Tip 8 trays per pack

Polyacrylamide HLT Flat Gel 10 10 up to 0,4 0,37 N/A x 200 tips per tray/ 732-2338sequencing gels 1 tray per pack

DNA sequencing gels HLT Ultra Flat Gel 10 10 up to 0,2 0,17 N/A x 200 tips per tray/ 732-23391 tray per pack

372-1054732-2339732-1055 732-2338732-1048732-2337732-2257732-2256732-2255

• Long, soft fine point will not endanger samples

• Simplicity of use relieves the frustration of ruinedexperiments

• Available in six styles as well as six OD’s

• Molecular BioProducts brand offers a complete lineof Gel tips ranging from 10-200 µl with ART selfsealing barrier


Procedure1. Autoclave (121 °C) Agar mix2. Cool down in a water bath (55 °C)3. Add Ampicillin-Na or Kanamycin stock solution to an end concentration of 100 µg/ml 4. Stir Agar mix on magnetic stir plate with stir bar to ensure homogenous antibiotics

distribution5. Setup Agar Mix to Omnispense Plus using 3 mm sized ID tubing for dispensing 25-30 ml

in a 10 cm petri dish6. Dispense Agar mix into a user-determined amount of 10 cm petri dishes.7. Let agar solidify at room temperature8. Transfer to 4 °C for short-term storage

ConclusionUsing the Omnispense Plus, distribution of agar into petri dishes saves significant time versusmanual pipetting. Set volumes will be continuously dispensed accurately without the hassleand time consumption of “re-filling”. Using the Omnispense Plus Automatic Dispenser forsimilar applications will prove to be time efficient, as well as cost effective.

The Wheaton Omnispense Plus is

an automatic dispenser that allows

for the dispersion of quantities of

set volumes. The Wheaton

Omnispense Plus is more accurate

and less time consuming than

manually pipetting. Dispensing

warm agar into petri dishes using

the Omnispense Plus is a proven

safe method.


Preparation of agar plates with antibiotics usingWheaton’s Omnispense® Plus

Agar recipe

• 10 g of NaCl • 10 g of Tryptone • 5 g of Yeast Extract • 20 g of Agar • Adjust pH to 7,0

(with 5 N NaOH if necessary) • Add deionised water to a final volume

of 1 l

Material

• Ampicillin-Na • Kanamycin • Water bath • Omnispense Plus automatic dispenser

(Cat. No. EU 224-0753; UK 224-0049 )• 10 cm petri dish • Magnetic stir plate with stir bar

FEATURES

• Ideal for packaging buffers,reagents and culture media

• Multi-language display-English(default), French, German,Spanish, Italian or Portuguese

• Operates as dispenser or a flowpump

• Use as stand alone dispenser or in-line filling pump

• Variable speed (0,34 to 29,1 ml per second

• Supports up to 3 pumpheads

• 4 tubing sizes to dispense 1 ml to 17,000 litres per dose

• Use in automatic or manual mode

• Autoclavable silicone tubing

• Store and retrieve 100programmes

• UL, CSA and CE compliant




Isolation of primary mouseembryonic fibroblasts (PMEF's)

Materials

• Phosphate buffer saline (PBS)• 10 cm tissue culture petri dishes• Potter-Elvehjem tissue grinder (Cat. No. 432-5047)• Trypsin/EDTA• DMEM + 10% FBS +1% pen/strep+ 1% L-glutamine• 680 cm2 glass roller bottle (Cat. No. 734-3021)• 490 cm2 plastic roller bottle• Roller Culture Apparatus (Cat. No. EU 734-1167; UK 734-1168)• Sterile Cryule® Vials (Cat. No. 216-3023)• Laminar flow hood.

Procedure1. Transfer tissue cells into a sterile 10 cm tissue culture petri dish with a sufficient volume of

Trypsin/EDTA to cover 2. Incubate the tissue for 15 minutes at 37 °C, and then grind the tissue a few times using a

Potter-Elvehjem tissue grinder (Cat. No. 432-5047) to dissociate the tissue. Allow the largepieces of cellular debris to settle. Remove the supernatant into a vessel and add 50 ml offibroblast media. Transfer to a 490 cm2 or 680 cm2 roller bottle. (Cat. No. 734-3021).Aliquot 7 to10 ml per bottle

3. Place bottles on Roller Culture Apparatus (Cat. No. EU 734-1167; UK 734-1168), Incubate at37 °C with 5% CO2. Roller speed should be set at a minimum until cells adhere to rollerbottle

4. PMEFs should attach and begin to divide in 1-3 days. After 2 days change the mediumwhich should be very acidic (indicated by the media turning yellow in colour).

5. When the bottles are confluent, usually in 3-4 days, the cultures are ready for freezing 6. Freeze cells in 10 % DMSO at 2 x 106 cells/vial using Sterile Cryule® Vials (Cat. No. 216-

3023)


Omnispense Plus Automatic Dispenser EU plug 224-0753Omnispense Plus Automatic Dispenser UK plug 224-0049

Dispense your timemore efficiently


Can I mix standard peptide andProjectPeptide in a single order?Yes. Just mention in your orderwhich peptides should be treatedas ProjectPeptides.

Can I order ProjectPeptide online? This feature is not yet available.As soon as it becomes available it will be communicated.

How do I order ProjectPeptide? Use our Excel ordering form that can be found atwww.thermo.com/peptides and [email protected].


NEW! ProjectPeptideTM Service4455%%

cchheeaappeerr!!

Standard Peptide Service ProjectPeptides Service

Manufacturing time 10 business days* 4 to 7 weeksQuality highest highestQC certificate analytical** HPLC + MS analytical** HPLC + MSModifications available unlimited limitedPurity crude to >99% crude to 95%Qantity 0,5 mg to 10 g and above 5 mg to 100 mgI.M.P. option yes noMaximum length >70aa max 25aa

Thermo Scientific is proud to introduce thenew ProjectPeptides™ service.

If you are able to plan your experiments a few weeks in advance ProjectPeptideswill contribute to maximise your budget use while getting the quality andservice you expect from us.

* Average for non modified peptides up to 97% purity** Not available for crude grade

Table1: Costs in Euros

purity 5 mg 10 mg 20 mg 50 mg 100 mg delivery time

Crude n.a. 6,11 8,16 12,24 16,31 4 weeks

70% 11,78 14,22 19,83 26,72 44,52 5 weeks

80% 15,18 18,07 23,34 32,82 52,74 6 weeks

90% 18,98 23,34 32,12 38,36 63,24 6 weeks

95% 22,78 28,61 40,84 44,08 70,32 7 weeks

ProjectPeptidesTM

• Maximum length 25aa• Minimum peptide price 6aa• Purities: crude - 95%• Limited modifications available,

please enquire • Prices in euros per amino acid (AA)

see table

>

FAQs


VWR Biosciences,more than just a helping hand



GGeennoommiiccss rroouunndd-uuppGenomics is the study of an organism's entire genome. The study of entire genomes is arelatively new field, which promises to unlock many secrets in biology. These include theidentification of disease causing genes, generation of more accurate evolutionary trees andunderstanding how cell responds to changes in its environment at a molecular level. All of thiswork is based upon our ability to align and sequence very large pieces of DNA to create acomplete map of each chromosome found in an organism.The importance of deoxyribonucleic acid (DNA) within living cells is undisputed. It is found in alldividing cells, largely if not entirely in the nucleus, where it is an essential constituent of thechromosomes. Many lines of evidence indicate that it is the carrier of a part of (if not all) thegenetic specificity of the chromosomes and thus of the gene itself. (J.D.Watson & F.H.C.Crick,Genetic Implications of the structure of deoxyribonucleic Acid, 1953).

DNA Analysis

Cogenics

Cogenics (formerly Lark Technologies and Genome Express) provides a comprehensive range ofmolecular and pharmaco-genomics services to pharmaceutical, biotech, academia, agriculturaland government clients. Cogenics currently runs and has in development numerous GLP-validated and research assays. These assays cover key SNPs relating to target identification, drugdevelopment activities and ultimately drug usage in specific populations and apply to bothbiologics and small molecules in all therapeutic areas. Cogenics has extensive experienceworking with platform providers including Affymetrix, Agilent, Sequenom®, ABI and others.

Services from Cogenics• Complete sequencing solutions for research, GLP, cGMP and CLIA-compliant services

are provided for any construct size or throughput• Primer Walking for DNA templates that require full length sequencing of a known /

unknown region• Genotyping for research, GLP, or CLIA-compliant DNA variation analysis. Services range

from whole genome scans to targeted SNP genotyping, using a variety of genotypingplatforms

• Microarry Services – providing both Affymetrix and Agilent platforms

Cogenics will soon be offering the next generation sequencing using the Roche GS-FLX. The 454Read Service will allow unprecedented throughput, sensitivity and reliability for a range of highthroughput sequencing applications, including, but not limited to:• Microbial whole genome sequencing• BAC sequencing• Metagenomics• Mutation discovery• miRNA discovery• cDNA library screening

Cogenics interacts with customers to provide solutions tailored to specific requirements. Inparticular, Cogenics is able to deliver custom bioinformatics packages to help clients maximisethe potential of their data. Because project management and data analysis are criticalcomponents of every next generation sequencing project, a dedicated project manager will beassigned to each project.

BioSciences

VWR BioSciences works with selectedpartners to help you drive on your businessin various fields including Genomics,Proteomics, Pre-Clinical, GLP or non-GLPstudies. This selection of strategic partnershas been carefully screened and selected byVWR, validating their capabilities, reliabilityand security on issues such as intellectualproperty. Our aim quite simply to provide thehighest quality services and hence eliminatethe researchers' need to spend countlesshours searching for a reputable firm. In thelast VWRbioMarke magazine we reviewedthe capabilities of VWR BioSciences in theincreasingly important proteomics field.In this edition we examine the genomicsportfolio of services ranging from DNA and RNA analysis to library formation.

>> new



RNA Analysis

RNAx

RNAx is an independent functional genomics research and service company based on theapplication of RNA interference. RNAx offers the application of RNAi technologies within the fullrange of the target discovery validation process, all the way from target identification, to pathwayelucidation and the generation of transgenic and knockout animals, based on RNAi technology.

ServicesValidation of SiRNAs and shRNAs – Test the ability of your si-RNA oligos, whether they are ableto knock down a target, measured by real time PCR. This includes efficiency test, titration studies,adaptation of cell lines, establishment of transfection criteria, and duration of knock down (knockdown experiments define the function of a target in a rapid, robust, and automated way).Validation of Targets in cell line – RNAx designs si/shRNAs and performs a knock down of thecustomer’s targets. A wide variety of human cell lines and primary cells are available for these studies.Using an automated system to seed, transfect the cells, and also perform real time PCR, achieves ahighly consistent and reproducible results. Several functional assays are available on the automatedplatform to check for phenotypic changes after mRNA knock down.

DNA/RNA oligo-synthesis

Thermo Biopolymers

Thermo Biopolymers has been producing reverse phase HPLC purified oligonucelotides since 1995.This method guarantees a constant, reliable and reproducible high purity for all oligonucleotides. Weare updating our portfolio continuously, so we are able to provide molecules required by scientificdevelopments including customised solutions. Every day 30% of the oligonucleotide production iscontrolled by mass spectrometry and analytical HPLC. To maximise the synthesis success rate ThermoBiopolymers have put in place preventive maintenance programs.

Services Primers, long oligos (up to 140 bases), with an extensive choice of modifications, LNA, RNAi,antisense oligos, oligo-enzyme-conjugates and dyes. All oligos are HPLC-purified (PAGE on request)and lyophilised, the products are delivered in 2-4 days after receipt of your order. Dyomics offersmore than 50 fluorophores covering the whole fluorescence spectrum. Coupled to oligonucleotidesthey are useful in fluorescence assays like FISH, Realtime PCR or FRET assays and they may be anattractive alternative for traditional fluorescence dyes.

VWR offer the most up to date and easy to use custom oligo configurator onwww.vwr.com simply click onto the bioSciences sections and then you will be guidedto it.

Gene synthesis

GenScript

GenScript's gene synthesis technology produces custom genes de novo using only the genesequence.

ServicesIn contrast to oligo synthesis, which can only create single-stranded DNA, GenScript's proprietarygene synthesis process routinely produces double-stranded synthetic genes of 10,000 bp or longer.The synthetic gene can be subcloned into any destination vector and is ready for use without furthersubcloning.

>>unique



For more information on these products contact your local VWR sales office,send an e-mail to [email protected] or visit our website www.vwr.comBioSciences

GenScript gene synthesis comes with complimentary codon optimisation, mRNA secondary structuremodification, and other procedures to enhance gene expression.Synthetic genes will be confirmed base-by-base using automated bi-directional fluorescent DNAsequencing and an 100% match with the requested sequences is guaranteed.

Transcriptome Services

Vertis Biotechnologie AG

Vertis Biotechnologie AG aims to bring the most efficient state-of-the-art tools for analysis of thetranscriptome to the worldwide biotech community. Their innovative technologies for geneexpression analysis and for gene discovery projects are listed below.

ServicescDNA synthesis and cDNA library construction – choose between four different techniques tosynthesise cDNA.Full-length gene cloning – isolation and cloning of full-length cDNA using its true full-lengthcDNA synthesis technique in combination with its unique method to clone uncut full-length cDNA.This method avoids treatment of the cDNA with restriction enzymes allowing cloning of full-lengthcDNA without loss of information due to the presence of internal restriction sites.Cloning and analysis of small non-coding RNA (e.g. miRNA, siRNA, ...) – a very reliable andsensitive method to synthesise cDNA from sncRNA (e.g. miRNA, siRNA). The resulting cDNAsrepresent valuable sources for the rapid experimental discovery of naturally occurring endogenoussmall RNAs.Profiling of gene expression using 'Massively Parallel Signature Sequencing' (MPSS) technology.

Genomic servicesGenomic library construction – VERTIS constructs libraries from nuclear DNA isolated from cellsor tissues. The genomic DNA is size fractionated and the range of 15-25 kb is cloned into the Bam HIrestrictions sites of Lambda phage vector LambdaGEM-11.DNA subcloning service – Cloning steps are often a central part of research projects, thesetechniques can be difficult and very time consuming to do in the research lab.

Custom BAC libraries

Amplicon Express

Amplicon Express is a provider of high-quality BAC Libraries and related services with thousands ofsatisfied clients all over the world.The BACMAPS (BAC Minimally Amplified Pooling Strategy) system enables researchers to use PCRfor screening a BAC library constructed from an organism with a very large genome. Since theexpense of picking millions of clones is cost prohibitive, Amplicon have developed a system thatallows screening of millions of clones with a few (48-384) PCR experiments and then following a hitto another plate and screening that DNA by a second round of 96 PCR reactions. This then identifiesa well that contains the BAC clone of interest as well as other BAC clones. The exact plate and wellposition containing the BAC clone of interest; together with 25-35 other BAC clones (or up to 80 inspecial cases) can be correctly identified in 192 PCR experiments. Following identification of thespecific plate and well position, the specific well is completely thawed, mixed thoroughly and platedon selective media to pick individual clones. Plating, picking and screening 96 white clones issuggested if the well pools have 35 or fewer initial clones and 384 clones if the well pools havemore than 50 clones.

Each kit is custom built for the researcher and the total number of MAPS Superpools in the kit willdepend on the total number of BAC clones in the library. Each MAPS Superpool will have acorresponding 96-well plate of MAPS clones. Each 96-well plate will have about 3,500,000 bp ofBAC clone inserts per well that corresponds to between 25 and 90 BAC clones (depending on theinsert size) pooled in each well.


Your European Distribution Partner

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Fax: 0 825 02 30 35 (0,15 EUR TTC/min)

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SpainVWR International Eurolab S.L.Apartado 4808100 Mollet del Vallés -BarcelonaTel.: 902 222 897Fax: 902 430 657E-mail: [email protected]

SwedenVWR International ABFagerstagatan 18a163 94 StockholmTel.: 08 621 34 00Fax: 08 621 34 66E-mail: [email protected]

SwitzerlandVWR International AGLerzenstrasse 16/18 8953 DietikonTel.: 044 745 13 13Fax: 044 745 13 10E-mail: [email protected]

UKVWR International LtdCustomer Service CentreHunter BoulevardMagna ParkLutterworthLeicestershireLE17 4XNTel.: 0800 22 33 44Fax: 01455 55 85 86E-mail: [email protected]

Special offers for life scientists in the VWR bioMarke Shop!

Molecular biology

• VWR Collection Unocycler and geldocumentation system

• DNA extraction kits from 5 PRIME• CBS PCR workstations• BTX HT Multi-Well Electroporation • Axygen plates for Real-Time PCR

and sequencing• RNase AWAY® or DNA AWAY® Wipes• Thermo Scientific Taq Polymerase

Cell Biology

• Optimise your immunoassays• Nunc OptiCell®• VWR Collection tube rotator• Microplate readers and washers from

Thermo Scientific

Proteomics

• ProteoExtract® Subcellular ProteomeExtraction Kit from Merck Biosciences

• Pall Acrosep Chromatography columns• Whatman FAST® slides for protein

microarray

Lab supplies

• New BD FalconTM flip top tubes • Brand Transferpette S multichannel• Nalgene tank Liners• Spectrum Mediakap

Biosciences

• Thermo Biopolymers ProjectPeptidesTM

• BioGenes monoclonals• Biosirius Protein Investigator


SSUE INTER vwr

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