issue 21 autumn 2008 -...

Issue 21 Autumn 2008

vwr.com

The testing of a new generation tissue homogeniser using bead beating technology

Bertin p. 22

BRANDplates® - the new range of microplates for cell culture

Brand p. 8

Multiskan FC - filter based microplate photometer for research

and routine applications

Thermo Scientific p. 16

Tangential flow filtration for small sample volumes as low as 2 ml with the KrosFlo® Research II TFF System

Spectrum p. 35

VWR I n t e r n a t i o n a l VWRbioMarke Issue 21 S e p t e m b e r 2 0 0 82 VWR I n t e r n a t i o n a l VWRbioMarke Issue 21 S e p t e m b e r 2 0 0 8 3

Innovation, Evolution, ExpertiseAs the year advances into autumn we hope that that you will appreciate this latest edition of the VWRbioMarke magazine - another bumper issue packed full of innovations and ideas.The creativity and scientific ingenuity of our VWRbioMarke partners can be seen from the evolution of Brand’s new surface coated plates to automated extraction of high quality DNA by a new bench top system from Mole; Microarray technology from CapitalBio and the detection of the phosphorylation status of key proteins disease related pathways from Merck. Indeed from cell biology to proteins and molecular biology plus helpful developments in liquid handling and sample preparation – they are all here!Included with this issue of the VWRbioMarke magazine is the VWRbioMarke shop, the tabloid filled with special offers on key products – often linked to the magazine articles. This year everyone is feeling the pinch of rising prices and spending restrictions so make sure that you have a look through this flyer to help you get the most out of your budget! There will be another edition in November.

Very best regards The VWRbioMarke Team

P.S To make sure that you get all the news about these and other new items as well as special offers and new catalogues register on our website to get the VWR e-newsletter.

Air Liquide

Applichem

BD Biosciences

Bertin Technologies

Biovest International

Brand

BTX Harvard Apparatus

CapitalBio

C.B.S. Scientific

Lonza

Merck Biosciences

Mole Genetics

Nalgene

Nunc

Omega Bio-Tek

Operon

Pall Life Sciences

5 PRIME

Quanta Biosciences

Sartorius Stedim Biotech

Spectrum Laboratories

Thermo Scientific

Thermo Biopolymers

VWR Collection

Wheaton Science Products

editorial

table of contents

EditorVWR International Europe bvbaHaasrode Researchpark Zone 3Geldenaaksebaan 4643001 LeuvenBelgium

CopywritingVWR International Europe bvba

Layout and typesettingMarketing Services VWR

PrintingStork, Bruchsal, GermanyNo part of this publication may be reproduced or copied without prior permission by writing of VWR International Europe.

Run72 250 copiesPublication date: September 2008

Due to the high sales volume of promoted articles some items may be temporarily out of stock - VWR Terms and Conditions of Sale apply.

Cell biologyWheaton – Using staining dishes and Colombia jars for demonstration of glycogen using periodic acid Schiff stain . . . 3BD – Provide your cells with the best environment using BD Matrigel™ Basement Membrane Matrix . . . . . . . . . . . . . . . . 4Lonza – ProNS0™ Protein Expression Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Brand – BRANDplates® - the new range of microplates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Biovest International – AutovaxIDTM: advanced hollow fibre bioreactors with automated lactate control yield higher density monoclonal antibody production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Nunc – Advantages of Nunc Bank-it and Cryobank for Biobanking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12Nunc – Nunc introduces the new EasyFill™ Cell Factory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Liquid handlingThermo Scientific – Improve sample precision and accuracy with low retention pipette tips . . . . . . . . . . . . . . . . . . . . . . . . . 15Thermo Scientific – Filter-based microplate photometer for research and routine applications . . . . . . . . . . . . . . . . . . . . . . . 16Thermo Scientific – Finnpipette F1 antimicrobial feature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Molecular biologyMole – GeneMole® automated system: Reproducible extraction of high quality DNA from 1-16 blood samples . . . . . . 18Applichem – New insights into DNA decontamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20Bertin Technologies – The testing of a new generation tissue homogeniser . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22CapitalBio – A comparison of static and 3D-rotation methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24Omega bio-tek – Isolation of DNA from blood samples and body fluids using E-Z 96TM Mag-Bind Blood DNA System . 26Thermo Scientific – Fluorescence Dyes – broadest portfolio and many new alternative dyes . . . . . . . . . . . . . . . . . . . . . . . . . 28Thermo Scientific – Quantitative 16S Ribosomal DNA detection using ABsolute QPCR master mix for diagnosis of central vascular catheter associated bacterial infection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305 PRIME – It´s time for 5 PRIME´s - PerfectPure RNA purification kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32BTX – phiC31 Integrase confers genomic integration and long-term transgene expression in rat retina . . . . . . . . . . . . . . 34

Sample preparationSpectrum – Tangential flow filtration for small sample volumes as low as 2 ml with the KrosFlo® Research II TFF System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35Pall Life Sciences – AcroPrep filter plates for efficient sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

ProteinsMerck – WideScreen™ Multiplex Assays for the Luminex® xMAP® Technology platform . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

ElectrophoresisCBS Scientific – 3 Different instruments for Mutation Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42


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

OverviewTissue sections are first oxidised by periodic acid. The oxidative process results in the formation of aldehyde groupings through carbon-to-carbon bond cleavage. Free hydroxyl groups should be present for oxidation to take place. Oxidation is completed when it reaches the aldehyde stage. The aldehyde groups are detected by the Schiff reagent. A colourless, unstable dialdehyde compound is formed and then transformed to the coloured final product by restoration of the quinoid chromophoric grouping.The PAS stain with diastase or -amylase digestion has histochemical specificity for glycogen. Skeletal muscle normally contains glycogen and is often recommended as a positive control tissue.

Equipment• 10-20slidestainingdish–WheatonScienceProducts(Cat.No.720-0701)• Columbiajar–WheatonScienceProducts(Cat.No.720-1035)• Coplinstainingjar–WheatonScienceProducts(Cat.No.631-2509)• Forceps• Latexgloves

Staining procedure 1. Place the cover slip with section in a Columbia staining dish (Cat. No. 720-1035). 2. Add Carnoy’s fixative to dish for 10 minutes. 3. Rinse very carefully with several exchanges of deionised water. Use caution, sections may rinse off. 4. Add periodic acid solution to staining dish for 10 minutes. 5. Rinse very carefully with several exchanges of deionised water. Use caution, sections may rinse off. 6. Add Shiff reagent for 5 minutes 7. Carefully wash with three exchanges of tap or deionised H2O. 8. Dehydrate in ascending alcohol solutions (50%, 70%, 80%, 95% x 2, 100% x 2) in 10-20 Slide staining dish –

Wheaton Science Products (Cat. No. 720-0701). 9. Clear with xylene (3 - 4 x) in Coplin Staining Jar (Cat. No. 631-2509). 10. Mount cover slip onto a labelled glass slide with Permount or comparable suitable organic mounting medium

ConclusionThe variety of Wheaton staining dishes allows flexibility for multiple staining applications. Wheaton staining dishes, covers, and racks are manufactured from soda lime glass and are available in many configurations to accommodate up to 50 slides, with various glass and stainless steel racks.

Using Wheaton staining dishes and Colombia jars for the demonstration of glycogen using periodic acid Schiff stain

Cell biology

Solutions: I. Carnoy’s fixative (store at room temperature) PREPARE IN A FUME HOOD Alcohol, 100% 60 ml Chloroform 30 ml Glacial acetic acid 10 ml

2. Periodic acid solution, 0,5% (w/v) PREPARE FRESH FOR EACH STAIN Periodic acid 50 mg dissolved in deionised water 10 ml

3. Alcohol 50% Reagent alcohol ~50 ml Deionised water ~50 ml



6. Alcohol 95% Reagent alcohol ~95 ml Deionised water ~ 5 ml

Reagents: • Absolutealcohol(100%ethanol)• Glacialaceticacid• Amylase

• Chloroform• Periodicacid• Permount

• Reagentalcohol,ACS• Schiffreagent• Xylene

Wheaton offers a variety

of staining dishes and

Columbia jars suitable

for a variety of histology

staining methods. Routine

staining is performed to

give contrast to the tissue

being examined as without

staining it is very difficult

to see differences in cell

morphology. Periodic acid

Schiff stain is used for the

demonstration of glycogen.

VWR I n t e r n a t i o n a l VWRbioMarke Issue 21 S e p t e m b e r 2 0 0 84

The basement membrane

Basement membranes are highly specialised, continuous sheets of extracellular matrices (ECM) that underlie epithelial and endothelial cells and surround muscle, fat, and the entire nervous system. Providing a mechanical support for cell layers, basement membranes furthermore play a key role in diverse biological processes. They direct the formation of barriers between tissue compartments, impeding the transmigration of cells and passively regulating the exchange of macromolecules. They also serve as interactive

surfaces for cells, actively providing signals for cell shape, adhesion, and migration, as well as communicating information for regeneration and/or differentiation.1 Basement membranes are composed primarily of laminin, collagen IV, heparan sulphate proteoglycan (percelan), and entactin.

BD Matrigel™ Basement Membrane MatrixBD MatrigelTM Basement Membrane Matrix is a solubilised basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma, a tumor rich in ECM proteins. Its major component is laminin, followed by collagen IV, heparan sulphate, proteoglycans, entactin, and nidogen.2 It also contains TGF-ß, fibroblast growth factor, tissue plasminogen activator3, and other growth factors that occur naturally in the EHS tumor. At room temperature, BD MatrigelTM Matrix polymerises to produce biologically active matrix material resembling the mammalian cellular basement membrane.

Promotes various cellular functionsBD MatrigelTM Basement Membrane Matrix promotes the attachment and differentiation of both normal and transformed anchorage-dependent

epithelioid and other cell types. These include neurons, hepatocytes, Sertoli cells, mammary epithelial, melanoma, vascular endothelial, thyroid, and hair follicle cells.4-7 BD MatrigelTM Basement Membrane Matrix was shown to influence casein gene expression in mouse mammary epithelial cells.8-9 It was also shown to support in vivo peripheral nerve regeneration 10 and to facilitate differentiation of bovine oviduct epithelial cells.11

Available in different formatsThe Growth Factor Reduced (GFR) product is useful as an alternative to BD MatrigelTM Matrix in many of the applications listed above where a more highly defined basement membrane preparation is desired. It has been used to define the signals necessary in the formation of canicular cell processes in bone cells,12 to elucidate the role of growth factors in the formation of tubules by primary mouse kidney cells,13 and for gene expression studies in primary mouse mammary epithelial cells.14 The phenol red-free product is suitable for assays which require color detection (e.g., fluorescence).

The described BD MatrigelTM Matrix variants are also available in a High Concentration (HC) format. Typical protein concentrations are between 18-22 mg/ml. BD MatrigelTM Matrix HC can be used to assess in vivo angiogenic activity of different

BD Matrigel™ Basement Membrane Matrix - the best environment for your cells, guaranteed LDEV-free

Human Submandibular Gland (HSG) cells cultured on GFR BD MatrigelTM Matrix differentiate to form acinar structures within 24 hours. The acini shown above were stained with H/E at 72 hours. Indirect immunofluorescence staining also revealed the salivary gland-specific cysteine protease inhibitor, cystatin, in HSG cell acini (data not shown). (Photo courtesy of Dr. Hynda Kleinman).

When choosing BD Matrigel™ Matrix, you get:

• A substrate promoting differentiation of many cell types

• After gelling, a three-dimensional basement membrane model

• An increased rate of human tumor cell growth in nude mice

• A choice of models to measure invasive potential of tumor cells

VWR I n t e r n a t i o n a l VWRbioMarke Issue 21 S e p t e m b e r 2 0 0 8 5


compounds by subcutaneous injection into mice (BD MatrigelTM Plug Assay).4-8 The high protein concentration augments the growth of tumors and also allows the BD MatrigelTM plug to maintain its integrity after injection. This keeps the injected tumor and/or angiogenic compounds localised for in situ analysis and/or future excision. A similar approach can be used for the transplantation and investigation of human tumour cells. Reports include studies with prostatic, breast, small cell lung, and colon tumour cells, as well as adrenal carcinoma, melanoma, and lymphoblastic leukemia cells.15- 20

A variety of applicationsInvasion assaysBD MatrigelTM Matrix can be used as a reconstituted membrane that provides a biologically active ECM for in vitro invasion assays. The enzymatic

degradation of type IV collagen by the cells has been strongly implicated in the invasion process. 21-22 It is the basis for several types of cell invasion assays (e.g., tumour invasion, or endothelial cell invasion for angiogenesis studies). 23-26

Metabolism/toxicology studiesBD MatrigelTM Matrix can also be used to construct in vitro models of liver cells for drug toxicity studies. It has been shown to suppress hepatocyte growth and prevent growth-associated dedifferentiation,27 as well as maintain liver-specific functions in vitro longer than most collagen-based systems.28-31

Guaranteed LDEV-freeRecently, it was discovered that the EHS sarcoma cell line can be contaminated with a virus named lactate dehydrogenase elevating virus (LDEV). LDEV only infects murine macrophages, and is not a risk for humans. Certain in vivo applications, however, can potentially affect the health of mice in animal breeding facilities (particularly if animals with an impaired immune system are infected).Using appropriate quality control processes, BD Biosciences has eliminated any risk of potential

viral contamination of the end product. BD Biosciences has made an effort to clear the source of BD MatrigelTM Matrix of LDEV particles and now certifies that all the BD MatrigelTM products, as well as some other ECM products, have been produced from an LDEV-

free EHS sarcoma line. BD Biosciences will continue to adjust its quality control processes to the latest scientific knowledge in order to assure that our customers always use the safest and most reliable product.

Cell biology

Scanning electron micrograph (SEM) of two human fibrosarcoma cells having digested the BD MatrigelTM Matrix occluding the membrane pore and migrating through the 8 µm pore of the PET membrane.

SEMs of hepatocytes cultured for two days on collagen I (1), collagen I gels (2), or BD MatrigelTM Matrix (3). Note the clusters of spherical cells for hepatocytes cultured on BD MatrigelTM Matrix, typical of differentiated cells.

References1. Haralson, M.A. and Hassell, J.R., Extracellular Matrix, A

Practical Approach. Oxford University Press, NY. 1995. 2. Kleinman, H.K., et al., Biochemistry 21:6188 (1982).3. McGuire, P.G. and Seeds, N.W., J.Cell.Biochem 40:215

(1989).4. Kleinman, H.K., et al., Biochemistry 25:312 (1986).5. Hadley, M.A., et al., J.Cell.Biol. 101:1511 (1985).6. Kubota, et al., J.Cell Biol. 107:1589 (1988).7. McGuire and Orkin, Biotechniques 5/6:456 (1987).8. Li, M.L., et al., Proc.Nat.Acad.Sci.USA 84:136 (1987).9. Barcellof, M.H., et al., Development 105:223 (1989).10. Madison, R., et al., Exp.Neurology 88:767 (1985).11. Streuli, C.H., et al., J. Cell. Bio. 115:1383 (1991).12. Vukicevic, S., et al., Exptíl Cell Research 202:1 (1992).13. Taub, M., et al., PNAS 87:4002 (1990)14. Joshi, M.S., J. Exp. Zool 260(2):229 (1991).15. Pretlow, T.G., et al., Cancer Research 51:3841 (1991).16. Noel, A., et al., Biochemical Pharmacology 43:1263 (1992).

17. Mehta, R.R., et al., Breast Cancer Res. and Treatment 25:65 (1993)

18. Fridman, R., et al., J. National Cancer Inst. 83:769 (1991).19. Sterling-Levis, K., et al., Cancer Research 53:1222 1(1993).20. Noel, A., et al., Anticancer Research 15:1 (1995).21. Liotta, L.A., et al., Cancer Metastasis Rev. 1:277 (1982).22. Melchiori, A., et al., Cancer Res. 52:2353 (1992).23. Taniguchi, B.M., et al., Cancer Research 49:6738 (1989).24. Terranova, V.P., et al., Proc. Nat. Acad. Sci. USA 83:465

(1986).25. Albini, A., et al., Cancer Research 47:3239, (1987).26. Hendrix, M., et al., Cancer Letters 38:137 (1987).27. Rana, B., et al., Mol. Cell. Bio. 14:5858 (1994).28. Schuetz, E.G., et al., J. Cell. Physiol. 134:309 (1988).29. Schuetz, J. D. and Schuetz, E.G., Cell Growth and Diff. 4:31

(1993).30. Kane, R.E., et al., In Vitro Cell Dev. Biol. 27A:953 (1991).31. Mann, D. J., et al., J. Mol. Endocrin. 8:235 (1992).

1 2 3

Ordering information see page 6


Product description Qty/Pk Cat. No.BD MatrigelTM Basement Membrane Matrix 5 ml 734-0270

10 ml 734-110050 ml (5x 10 ml) 734-0271

BD MatrigelTM Matrix High Concentration (HC) 10 ml 734-0273BD MatrigelTM Matrix, Phenol Red-free 10 ml 734-0272BD MatrigelTM Matrix, Phenol Red-free, HC 10 ml 734-1402BD MatrigelTM Matrix, Growth Factor Reduced 5 ml 734-0268

10 ml 734-0269BD MatrigelTM Matrix, Growth Factor Reduced, HC 10 ml 734-1441BD MatrigelTM Matrix, Growth Factor Reduced, Phenol Red-free 10 ml 734-1101BD MatrigelTM hESC-qualified Matrix (for feeder-free maintenance of human Embryonic Stem Cells)

5 ml 734-1440

BD™ Dispase 100 ml (50.000 caseinolytic units)

734-1312

BD Cell Recovery Solution 100 ml 734-0107BD BioCoat MatrigelTM Matrix Multiwell Cellware 6 well 2 734-0128

12 well 2 734-016924 well 2 734-012948 well 2 734-0173

BD BioCoat MatrigelTM Matrix Culture Dishes, 35 mm 8 734-0145BD BioCoat MatrigelTM Matrix Thin-Layer Multiwell & Assay Plates,

6 well 5 734-023424 well 5 734-105696 well 5 734-0235

BD BioCoat MatrigelTM Matrix Thin-Layer Culture Dishes, 35 mm 20 734-023360 mm 20 734-0232100 mm 20 734-0231

BD BioCoat MatrigelTM Matrix for Hepatocytes, 6-well plates 5 734-1050BD BioCoat MatrigelTM Matrix for Hepatocytes, Culture Dishes, 100 mm 5 734-1064BD BioCoat MatrigelTM Matrix for human Embryonic Stem Cells, 6-well plates

5 734-1404

BD BioCoat MatrigelTM Matrix Cell Culture Inserts, 0,4 µm inserts

in four 6 well plates 24 734-1046in two 24 well plates 24 734-0135

BD BioCoat MatrigelTM Matrix Invasion Chambers, 8,0 µm inserts

in four 6 well plates 24 734-1048in two 24 well plates 24 734-1047

BD BioCoat Growth Factor reduced MatrigelTM Matrix Invasion Chambers, 8,0 µm inserts in four 6 well plates

24 734-1049

BD BioCoat Growth Factor reduced MatrigelTM Matrix Cellware, 24 well plates

5 734-1065

BD BioCoat Angiogenesis System: Endothelial Cell Invasion, 24 well insert system

1 734-10185 734-1019

BD BioCoat 24 Multiwell Tumor Invasion System (8,0 µm pore size) 1 734-10245 734-1025

BD BioCoat 96 Multiwell Tumor Invasion System (8,0 µm pore size) 1 392-25005 392-2501

Ordering information



BioWhittaker® ProNS0™ 1 and 2 Chemically Defined, Protein-free Media, together with ProNS0™ Lipid CD supplement, are designed to meet a growing demand for optimised NS0 formulations.

BioWhittaker® ProNS0™ Media • Twooptimisedformulasareavailabletocover

the broadest range of NS0 nutritional needs • Furthermaximiseproteinproductionbytitration

with ProNS0™ Lipid CD supplement • Protein-freeandchemicallydefinedformulations• Productpurificationissimplified• Lot-to-lotconsistencyensuresdependable

performance • Nonanimalorigincomponentsreduceregulatory

burdens

This trio of NS0 products provides the answer to maximal protein productivity!

ProNS0™ Chemically Defined, Protein-free Media Two distinct formulae are offered to cover a broad range of nutritional needs. Titering the separate lipid supplement permits further optimisation that can have a significant positive impact on protein production. Suggested starting concentration for optional addition of ProNS0™ Lipid CD Supplement is 5 ml per litre.

Comparative protein production data are presented in Figure 1. All media were prepared according to

manufacturer instructions. ProNS0™ Media were supplemented with 5 ml per litre of ProNS0™ Lipid CD. Competitive media either contained lipid or were supplemented with manufacturer’s companion lipid according to manufacturer recommendations. The batch cultures were not fed or further supplemented.

Samples were taken daily to measure viable cells and IgG concentration. Cell counts and viability were measured using a Vi-Cell™ viable cell analyser (Beckman Coulter). IgG concentrations were measured by HPLC.

The data show that both ProNS0™ 1 and 2 Chemically Defined, Protein-free Media yield superior protein production compared to competitive media.

Duplicate 125 ml shaker flasks were seeded at a density of 200,000 NS0 cells per ml in a 30 ml volume. Shake rate was 100 rpm. Cells were cultured in their respective test media for one passage prior to test initiation.

ProNS0™ Protein Expression Media

Cell biology

Description Pack Cat. NoBioWhittaker® ProNS0™ 1 Chemically Defined, Protein-free Medium 1 l 733-1788BioWhittaker® ProNS0™ 2 Chemically Defined, Protein-free Medium 1 l 733-1789BioWhittaker® ProNS0™ Chemically Defined Lipid Supplement with cholesterol 5 ml 733-1790

The NS0 cell line is widely used for recombinant mammalian protein expression. Some of the reasons to select this mouse myeloma platform are:

– Forms stable producing hybrid cells with high levels of protein production – Lacks ability to secrete endogenous antibody or antibody fragments – Resists aggregation clumping in suspension culture – Lacks adverse protease activity on recombinant protein product (in many cases)

Figure 1. Protein production - ProNSOTM Media


Non-treated polystyrene (PS) is hydrophobic and leads to a very low cell attachment and spreading. Adherent cells prefer polar

surfaces that possess hydrophilic functional groups.

The BRANDplates® cellGradeTM line perfectly matches this demand. The surface modifications of the BRANDplates® are generated via a physical/chemical process, each with distinctive parameters. Different parameters modify the PS surface incorporating hydroxyl-, carboxyl-, oxygen- and amino-groups on the PS structure.

All BRANDplates® microplates can be stored at room temperature. All microplates for cell culture applications are sterile according to Ph. Eur. and USP29, free from endotoxins, DNase, DNA, RNase and non-cytotoxic.

Conclusion: The growth of HepG2 cells on the cellGrade™ plus surface was compared with high quality TC surfaces from two competitors. HepG2 cells showed an equivalent cell growth on the three different TC surfaces. Cell adhesion was significantly better on cellGrade™ plus surface.

Cell culture is experiencing increasing popularity in the research and development area. However, even outside of basic research, cells are cultivated today for a number of reasons, including the production of proteins and in particular as assay systems. As cell cultures can sometimes be quite demanding regarding their environment, the disposables used for cultivation have to be of highest quality.

BRANDplates® The new range of microplates!

Part 1: BRANDplates® microplates for cell culture – select the best surface for your cell line!

See our offer in

Figure 1. Proliferation assay with HepG2 cells.

Figure 2. Adhesion assay with HepG2 cells.


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

Please visit www.vwr.com and use the BRANDplates® selection guide for selection and catalogue numbers.

cellGrade™For the standard cultivation of adherent cell cultures- PS surface with different chemical

groups, like e.g. carboxyl and hydroxyl groups, that are freely accessible

- Surface is hydrophilic compared with untreated PS - Serum components are easily bound onto the freely

accessible chemical groups, allowing an indirect adhesion of cells

cellGrade™ plusFor serum-reduced media cultivation of fastidious cells- In addition to carboxyl and hydroxyl functional groups, free amino groups are present on the surface- The surface has a protein-like character; cells can directly attach and spread out- Especially suited for serum-reduced and serum-free cultivation of cells

cellGrade™ premiumPoly-D-Lysine-equivalent surface for cultivation of cell lines with the highest demands- Poly-D-Lysine-equivalent surface, with analogous results regarding growth performance and cell

morphology- High degree of amino-groups on the surface- Optimal adhesion of cells to the surface reduces cell damage when washing frequently- Surface suited for serum-free and serum-reduced cultivation of cells

BRANDplates®

for cell culture

Figure 3. HepG2 cells growing on cellGrade™ plus surface.


AutovaxIDTM: advanced hollow fibre bioreactors with automated lactate control yield higher density monoclonal antibody production

The AutovaxIDTM, as shown in figure 1, utilises modern hollow fibre technology for growth and production from both suspension and

anchorage-dependent cells lines. The AutovaxIDTM mimics mammalian physiology to create an optimal environment for cell growth and production. For example, the hollow fibres act similar to capillaries, the circulation pump mimics the heart, the gas exchanger serves as lungs and the AutovaxIDTM embedded process controller as the brain. Utilising EC CYCLINGTM, a process that alternates flow of media evenly between the lumen and exterior of the hollow fibres, media is efficiently mixed to reduce microenvironments and gradients within the bioreactor. This advance creates an optimal culture environment leading to a healthy and productive bioreactor. Throughout the culture duration, secreted product is continuously harvested into the built-in refrigeration unit.

This system was designed for cost-effective use, by nearly eliminating the need for technician interaction or expertise typically required for optimal high-density perfusion culture. As a result, personnel time is more available for other projects and not spent labouring over issues associated with large-scale cell culture. Here we present three examples of monoclonal antibody production with the AutovaxIDTM. By comparing variables typically associated with large-scale culture, the

AutovaxIDTM is a cost effective way to produce gram quantities of these products.

IntroductionThe AutovaxIDTM is an advanced, more automated, instrument compared to established ACUSYST® hollow fibre technology. For example, cultureware is completely enclosed, completely disposable, incorporates a pre-sterilised pH probe, and has factor, feed and waste pumps preloaded into a single cassette. By incorporating these advances, the set-up time has been reduced from several hours to several minutes. The system’s software prompts each step in process from fill & flush through inoculation. Once the cells are in the bioreactor, the system continually monitors lactate concentration and automatically adjusts medium delivery rates to accommodate optimal expansion of the cell population. The system notifies the technician when media and harvest containers need to be changed and when pH or other variables drift from predetermined set points. Overall the system was designed to be a standard piece of laboratory equipment that requires little expertise or labour to produce gram quantities of monoclonal antibodies.

materials and methodsThe National Cell Culture Center, which provides antibody production services to the academic community, routinely uses the AutovaxIDTM as a bioreactor to satisfy requests for mid-range quantities (0,5 to 5 g) of monoclonal antibodies. Although these levels are not pharmaceutical scale, they do represent a need for large-scale production that is common in diagnostic and research organisations, yet beyond the available capacity of many individual laboratories. Production data are shown here from three murine hybridoma cell lines cultured in the AutovaxIDTM from 19 to 49 days. The perfusion media (intracapillary, IC) was Biovest Basal, which is a proprietary media designed to use in hollow fibre bioreactors. The extracapillary (EC) media was 5% FBS for cell line A, 10% FBS for cell line B, and HSFM (serum free media, Invitrogen) for cell line C. Cell lines A and C were mouse hybridomas and B was a rat hybridoma line. All three cell lines were created by individual investigators and sent to the National Cell Culture

Figure 1. AutovaxIDTM Instrument and

Cultureware

The AutovaxIDTM is an enhanced, fully enclosed, automated hollow fibre bioreactor system for production of mammalian cell secreted proteins.



Center for large-scale antibody production. Additionally, these three runs were terminated when the requested quantity of antibody was produced.

Each cell line was initially expanded in T-flasks prior to inoculation in the AutovaxIDTM 1,1 m2 bioreactor. The T flask secretion IgG levels for A, B, and C, were 40 µg/ml, 50 µg/ml, and 10 µg/ml, respectively. The inoculum for A, B and C was 2,8 x 108 cells, 1,5 x 108 cells, and 2,5 x 108 cells, respectively. Just prior to inoculation, a 20 litre bag containing Biovest Basal was attached to the AutovaxIDTM along with 3 litres of EC factor (5% FBS, 10% FBS or HSFM). By following brief software prompts to fill & flush, an initial pH calibration was performed and cells were inoculated into the bioreactor. After inoculation, the AutovaxIDTM automatically adjusted the media feed rate in response to the expanding

cell population. Through feedback control and indirect lactate determination, the AutovaxIDTM automatically adjusted the media feed rate to maintain a lactate setpoint of 1,0 gm/l, until the maximum pump rate was reached. At this point (approx. day 10-14), a slow, continuous pumping of harvest and EC media was initiated and maintained for the duration of each culture. For informational purposes, daily samples were drawn to determine glucose uptake and lactate production rates. These samples were not required to operate the AutovaxIDTM and only used for historical comparison to ACUSYST® instrumentation (data

not shown). Following inoculation, the system operated without technician interaction except when prompted by the AutovaxIDTM to replenish the medium feed source.

ResultsAs shown in Table 1, cell line A was cultured in the AutovaxID for 34 days and consumed 220 l of basal media and 3 l of EC media. A total of 2 210 mg of antibody (IgG) was harvested in a total volume of 3,7 l (0,6 mg/ml).

Cell line B was cultured in the AutovaxID for 24 days and consumed 200 l of basal media and 3 L of EC media. A total of 1 000 mg of antibody (IgG) was harvested in a total volume of 3,0 l (0,3 mg/ml).

Cell line C was cultured in the AutovaxID for 49 days and consumed 175 l of basal media and 3,7 l of EC media. A total of 5 600 mg of antibody (IgG) was harvested in a total volume of 3,02 l (1,9 mg/ml).

As detailed in figure 2, the AutovaxIDTM controlled the Glucose Uptake Rate (GUR) by controlling the Lactose Production Rate (LPR) and once stabilised, the two rates were controlled within a few percentage points of each other.

ConclusionThe AutovaxIDTM is an advanced hollow fibre bioreactor system for the production of monoclonal antibodies. The data presented here demonstrate that, depending on the cell line-specific productivity, the AutovaxIDTM is a cost-effective approach for producing gram quantities of monoclonal antibodies. The enclosed cultureware and simplified set-up were designed to minimise technician interaction and eliminate the possibility for mistakes. More importantly, automated control of the media feed rate led to optimal cell expansion and product harvest, without the need for expertise in high density perfusion culture methodology. Finally, concentrated, cell-free harvest (product) was produced with approximately 75% less labour than typically required with conventional hollow fibre systems. Overall, the AutovaxIDTM represents an improved hollow-fibre bioreactor technology for mid-range production of monoclonal antibodies that are suitable for research or GMP applications.

Cell biology

Table 1. Media consumption and IgG production from three different murine hybridoma cell lines in the AutovaxIDTM.

CellLine Growth IC Media EC Medial Harvest Total Period Consumed Consumed Volume Antibody (days) (L) (L) (L) Produced (mg)

Hybridoma A (mouse) 34 220 3,00 3,70 2210

Hybridoma B (rat) 26 200 3,00 3,00 1000

Hybridoma C (mouse) 49 175 3,70 3,02 5600

Figure 2. Glucose Uptake Rate (GUR) and Lactate Production Rate (LPR) from three different murine hybridoma cell lines in the AutovaxIDTM.


Advantages of Nunc Bank It and Cryobank for Biobanking

DNA researchers often use labware made of polypropylene (PP) to store samples for prolonged periods of time, which may be a total many years. Adsorption of DNA to the containing vessel decreases its sample concentration. PP is a hydrophobic polymer and will therefore inhibit adsorption of the very hydrophilic DNA molecule. However, it has been observed that DNA can adsorb to PP and also that there is a large difference in DNA adsorption for different PP grades and/or different surface treatments. For long-term and low-temperature storage of small amounts of DNA, in for example bio-bank, it is important to identify a polymer surface with low adsorption of DNA. We have analysed several PP resins/modifications and identified a PP modification that offers low DNA adsorption. The performance of the identified PP candidate has been compared to a number of different commercially available DNA containers manufactured from PP.

MethodTo avoid differences in surface/volume conditions when comparing containers from different manufactures, test samples (d=4 mm) were die cut from the containers. 50 µl DNA solution (4,5 ng non-labelled Lambda DNA and 0,5 ng 32P-labelled Lambda DNA) was dispensed into Nunc PP Modules (Cat. No. 735-0075) and the test samples were placed on top. PP has a density of approximately 0,9, and will therefore float on the liquid and only one side of the test sample will be exposed

to the DNA solution. The modules were sealed and incubated overnight. After incubation, the test sample was removed, washed in TE-buffer and radioactivity remaining on the test sample recorded by Packard Cyclone Phosphor Imager. In order to calculate the amount of DNA adsorbed to the test sample, a DNA standard curve was made based on 2-fold dilutions of the DNA solution. 8 µl was applied to reference test samples and after evaporation of the liquid, they were incubated and exposed together with test samples. DNA adsorption were tested under different conditions such as high and low ionic strength buffers, high and low DNA concentration and storage at –20 °C for 1 and 7 days, respectively. Preparing of test material Lambda DNA was digested with Hind III and end-labelled with α32P-dGTP using a Nick Translation Kit (Amersham). Labelled fragments were purified by ProbeQuantG-50 column (Amersham) and purified DNA quantified by measuring adsorption at 260 nm (Envision Instrument 2100). Standard procedures were used. High ionic strength buffer: 2,5 M NaCl in TE buffer. Low ionic strength buffer (TE buffer): 10 mM Tris HCl, 1 mM EDTA.

ConclusionFor storage of small amounts of DNA, it is important to identify a polymer surface with low adsorption of DNA. Test samples have been die cut from commercial available containers commonly used for DNA storage as well as from containers produced in-house from different PP grades and/or different surface treatments.

The Bank It and Cryobank systems are the second generation CryoTube™ systems from Nunc. The first CryoTube™ was originally developed by Nunc for the WHO in the 1960’s. The Bank It and Cryobank systems are similar, but, feature different surfaces for DNA and Cells/proteins, respectively. They are available in 0,5 ml and 1,0 ml versions. The Cryobank systems are used by a number of cell and biobanks throughout Europe, while the Bank It vials are used for DNA storage in for example forensic work and biobanking.

The system provides the user with

• Themostspacesavingcryovialsystemonthemarket

• 2Dbarcodingthatallowstheusertoscanracksoftubesandtherebyavoidaccidential misplacing of the tubes

• Theabilitytoautomatethedecappingwith8and96decappers,andutiliserobotic liquid handling in the 96 well rack

• Asetofaccessoriesthatallowsfastpickingofvialstominimisefreeze/thawcycles

Marwood Kristensen, Thermo Fisher Scientific, Denmark


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

Figure 1.- Autoradiogram of DNA adsorption to various polypropylene containers (Con.). The intensity and size of the spot is proportionally to the amount of adsorbed DNA. DNA was incubated in high ionic strength buffer at RT for 20 hours. DNA dilution (T3) was included with all exposures. For data analysis, a grid specifying a definite area around each test sample was applied and total DLU (Digital Lights Unit) counted.

0

0,05

0,1

0,15

0,2

0,25

0,3

Con.

1

Con.

2

Con.

3

Con.

4

Con.

5

Con.

6

Con.

7

Con.

8

Con.

9

Con.

10

Con.

11

0

0,01

0,02

0,03

0,04

0,05

0,06

0,07

Con.

1

Con.

2

Con.

3

Con.

4

Con.

5

Con.

6

Con.

7

Con.

8

Con.

9

Con.

10

Con.

11

0

200000

400000

600000

800000

1000000

1200000

Con.

1

Con.

2

Con.

3

Con.

4

Con.

5

Con.

6

Con.

7

Con.

8

Con.

9

Con.

10

Con.

11

0

50000

100000

150000

200000

250000

300000

350000

Con.

1

Con.

2

Con.

3

Con.

4

Con.

5

Con.

6

Con.

7

Con.

8

Con.

9

Con.

10

Con.

11

0

0,05

0,1

0,15

0,2

0,25

0,3

Con.

1

Con.

2

Con.

3

Con.

4

Con.

5

Con.

6

Con.

7

Con.

8

Con.

9

Con.

10

Con.

11

0

0,01

0,02

0,03

0,04

0,05

0,06

0,07

Con.

1

Con.

2

Con.

3

Con.

4

Con.

5

Con.

6

Con.

7

Con.

8

Con.

9

Con.

10

Con.

11

0

200000

400000

600000

800000

1000000

1200000

Con.

1

Con.

2

Con.

3

Con.

4

Con.

5

Con.

6

Con.

7

Con.

8

Con.

9

Con.

10

Con.

11

0

50000

100000

150000

200000

250000

300000

350000

Con.

1

Con.

2

Con.

3

Con.

4

Con.

5

Con.

6

Con.

7

Con.

8

Con.

9

Con.

10

Con.

11

0

0,05

0,1

0,15

0,2

0,25

0,3

Con.

1

Con.

2

Con.

3

Con.

4

Con.

5

Con.

6

Con.

7

Con.

8

Con.

9

Con.

10

Con.

11

0

0,01

0,02

0,03

0,04

0,05

0,06

0,07

Con.

1

Con.

2

Con.

3

Con.

4

Con.

5

Con.

6

Con.

7

Con.

8

Con.

9

Con.

10

Con.

11

0

200000

400000

600000

800000

1000000

1200000

Con.

1

Con.

2

Con.

3

Con.

4

Con.

5

Con.

6

Con.

7

Con.

8

Con.

9

Con.

10

Con.

11

0

50000

100000

150000

200000

250000

300000

350000

Con.

1

Con.

2

Con.

3

Con.

4

Con.

5

Con.

6

Con.

7

Con.

8

Con.

9

Con.

10

Con.

11

Figure 2. - Adsorption of DNA to test samples at high and low ionic strength buffer, respectively. Low values indicates low DNA adsorption. High ionic strength promotes binding to PP, however adsorption does also take place at low ionic strength buffer. Test samples were die cut from various PP containers and incubated in a solution of 32P-labelled DNA (0,1 ng/lµl).

Test samples incubated overnight at 20 °C, and radioactivity adsorbed to the test samples was measured. DLU = Digital Light Units. Con. = Container. Container 2 is die cut from cryotubes produced by Nunc with a PP modification. DNA adsorption in low ionic strength buffer to container 2 corresponds to 5% of the container with highest DNA adsorption.

Figure 3. - Incubation of test samples in high concentration DNA solution. Test samples were incubated in a solution of 32P-labelled DNA (0,4 ng/µl) dissolved in high and low ionic strength buffer, respectively. Samples were incubated overnight at 20°C, and radioactivity adsorbed to the test samples was measured.

A DNA dilution was included in the experiments and was used to calculate the amount of DNA adsorbed to the test samples. Container 2 is die cut from cryotubes produced by Nunc with a PP modification.

Figure 4. - DNA adsorption after 1 and 7 days of storage at –20 °C. Test samples were incubated in a DNA solution (0,1 ng/µl) in low ionic strength buffer for 1 and 7 days, respectively. A DNA dilution was included in the experiment and incubated under the same conditions as test samples. The DNA dilution was used to calculate the amount of DNA adsorbed to the test samples. Container 2 is die cut from cryotubes produced by Nunc with a PP modification. There is still a pronounced difference in DNA adsorption between the various

containers after 7 days of storage, however Container 2 is still among the containers with lowest DNA adsorption.

High ionic strength buffer Low ionic strength buffer

High ionic strength buffer Low ionic strength buffer

DNA absorbed after 1 day storage DNA absorbed after 7 days storage

14

Versatile and Easy to UseNunc™ introduces the new EasyFill™ Cell Factory

The EasyFillTM Cell Factory is a unique system with one large and one small opening in each unit, which makes it versatile and easy to

use. The EasyFillTM Cell Factory bridges small-scale research and large scale GMP production through its unique design: Use it as it is without any accessories or use the plug-and-play connections for rapid attachment of tubing and filters significantly reducing contamination risk. Nunc has more than 30 years of experience with Cell Factories and was the first of its kind out on the market. We have invested in this new design to ensure long-term quality and supply for customers that rely on our product.

Nunc EasyFillTM systems are 25 cm wide by 33,5 cm long and available with one, two, four or ten growth chamber levels. The four different models provide surface areas ranging from 630 to 6300 cm2. Constructed of optically clear polystyrene so you can directly observe cells using an inverted microscope. EasyFillTM are available with Nunclon ΔTM surface treatment to promote consistent performance for cell attachment and proliferation. Shipped sterile and ready to use, for single use applications.

Benefits of the EasyFillTM Cell FactoryVersatile – The large opening facilitates the desire to pour media directly and the small opening supports those that need a closed, aseptic system for filling and harvesting• Bridgessmallscaleprocessdevelopmentwithlarge

scale production• Easy-to-useandready-to-userightoutofthebox,

no accessories needed• Highyieldandprocessefficiencyinaspacesaving

cell culture device• Highlevelofvalidationsupportthroughextensive

documentation

Specific areas of interest for EasyFillTM would be academia, R&D, process development, pilot manufacturing, bio mass production for feeding into screening departments, and full scale (GMP) production. EasyFilTM Cell Factory complements an already extensive cell culture portfolio: EasyFillTM is a product within the broad portfolio of Nalgene and Nunc brand products covering Fluid Containment, Cell Culture, and Fluid Transfer to fulfil the upstream and downstream needs of bio processing applications, including automation.

We have identified a PP modification (container 2 in the figures) that has a remarkably low adsorption of DNA. Containers produced with this particular PP were tested against several commercial available containers used for DNA storage, some of which are specifically sold as being low-DNA binding. The PP resin identified by Nunc was in all experiments amongst the best performers. Under common storage conditions (low ionic strength buffer) these container shows a DNA adsorption corresponding to less than 5% of the container with the highest DNA adsorption (Figure 2). It was also investigated if low DNA adsorption was related to the amount of DNA incubated with the sample. However, Figure 3 shows that even at high DNA concentration the same tendency for DNA adsorption between the various PP containers is still observed. Long-term storage of DNA (7 days, -20 °C, Figure 4) does not change the picture. It is important to point out that the

PP modification identified here improves DNA recovery without the use of any additive or coatings that can interfere with the biological material.

Cryobank Low Binding PropertiesThe Bank It material is optimised for low DNA adsorption, and similarly the Cryobank material resembles the material used in Nunc CryoTubes and as such exhibit low adsorption of proteins and low adhesion of cells. Two layer assays with incubation of e.g. surplus IgG or AFP in neutral PBS followed by detection with HRP linked secondary antibody can be used to detect this effect.Most illustrative is, however the effect, when mammalian cells are incubated in the vials, the tubes are washed and the adhered cells are stained. This is shown in photo 1.

Photo 1. L929 Cell Adsorption to CryoTubes™ (3 days incubation)

Nunc CryoTubes™ exhibit low binding characteristics both in terms of cellular adhesion and protein binding when compared with competitive tubes.

Various competitors

NuncTM

VWR I n t e r n a t i o n a l VWRbioMarke Magazine Issue 21 S e p t e m b e r 2 0 0 8


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

Improve sample precision and accuracy with low retention pipette tips

Precision and accuracy are major factors of pipette tips. Sample retention and the unpredictability of this retention between tips

is a key issue that leads to inconsistent data. Hence the need for a low retention pipette tip. Decreased retention will increase analytical accuracy of many methods, such as PCR , and with reagents becoming more expensive it may be more cost effective to utilise the low retention pipette tips, depending on your specific testing.

The effectiveness of Low Retention pipette tips by Molecular BioProducts, a product offering of Thermo Scientific, compared to standard pipette tips, were examined in an independent laboratory study. The recovery of an oligonucleotide to determine the retrievability of pipetted samples was analysed and the binding of 32P labeled oligonucleotides to pipette tips was measured. The new low retention tips supported Molecular BioProducts’ claims by increasing sample recovery and performing more precisely than standard pipette tips.

As shown in Table 1, oligonucleotide HAV shows a mean CPM retention of 1451 CPM in the low retention tips. In the standard tips, 3611 CPM was observed. The results of HAV probe’s mean CPM using standard tips shows a 149% greater retention of the oligonucleotide HAV when compared with the mean CPM using the low retention tips. This means that the standard tip results in 2,5 times the retention of that seen with the low retention tips. This 1:100 solution left 11% of the total HAV 32P labeled oligonucleotides in the standard pipette tips as compared to 4,4% in low retention tips.

Perhaps more importantly, the low retention tips were more precise in the amount of oligonucleotide retention. The standard deviation in the amount of oligonucleotide held in the tips was just 684,8 CPM in the low retention tips versus 2336,4 CPM in the standard tips.

In determining which pipette tip to use in your laboratory, it is important to evaluate the level of precision and accuracy you need. The results of this experiment show that the low retention tips are more accurate in dispensing the sample pipetted, which should save on reagent costs and improve efficiency in your experimentation. The results also demonstrated that the low retention tips are more consistent in the amount of oligonucleotides that adhere to it. This will increase the precision of sample transfer, which should yield more consistent results in your experimentation.

Key Benefits:• Precisesampledelivery

• Savingsonexpensivereagents

• Morereliableresultsincritical applications

• Lessliquidretentionand enhanced liquid handling

• Contaminationprevention, certified RNase, DNase, ATP & Pyrogen free

Table 1. Summary of HAV probe results(Mean CPM is counts associated with tip after washing)

Diluted HAV probeLow Retention Standard

Mean CPM 1451,3 3611,0SD CPM 684,8 2336,4


Designed with 30 years experience in microplate photometry, the Multiskan FC combines the world renowned quality of the Multiskan product line with a large colour screen, visual internal software with ‘quick keys’ and multiple language options to ensure excellent usability.

Multiscan FC offers you

• Abroadwavelengthrangeof340-850nm for a wide variety of research and routine applications

• Fastandaccuratemeasurementofboth 96 and 384 well plates for varying throughput requirements

• Shakingandincubationupto50°Cfortemperature critical assays

• Easeofusethroughthelargecolourscreen, visual internal software and a variety of language versions

• VisualandlogicalSkanItSoftwareforcomprehensive instrument control and data handling

• Provenperformanceandreliableday-to-day results through patented optical design and in-built self diagnostics

• Designedwith30yearsexperienceinmicroplate photometry

Multiskan FC for a wide variety of photometric applications

The Multiskan FC boasts a 340 – 850 nm wavelength range, enabling a variety of applications from enzyme kinetic studies through to Lowry assays. It is equipped with an eight position filter wheel with three standard filters 405, 450 and 620 nm pre-installed. A comprehensive range of easy to install additional filters is available.

The Multiskan FC provides fast and accurate measurements providing complete 96 well plate reading in less than 6 seconds. The instrument is equipped with linear shaking as standard and an optional incubator can provide incubation temperatures up to 50 °C and the capacity to read 384 well plates.

Reliable results and robust performance The proven and patented optical design of the Multiskan FC in combination with the auto-calibration procedure that is performed during each measurement, guarantees stable day-to-day and year-on-year performance and reliability.

During start up all major functions of the instrument, such as plate position, measurement stability, lamp functionality, filters, optical system, incubation and electronic operation, are checked to ensure reliable operation. Additionally, the lamp is automatically switched off when not in use, thus prolonging the lifetime of the lamp.

Ease of use with internal softwareThe large colour screen of the Multiskan FC combined with the simple and logical internal software ensures easy and intuitive assay setup. The ‘quick keys’ allow instant

Thermo Scientific Multiskan FC – Filter-based microplate photometer for research and routine applications

The Thermo Scientific Multiskan® FC microplate photometer is a reliable and robust instrument for a wide variety of research and routine applications. It reads both 96 and 384 well plates, and is equipped with shaking as well as incubation capabilities for temperature critical assays. It can be used as a stand-alone instrument or under PC-control with our intuitive Thermo Scientific SkanIt® Software.

Description Cat. No.Multiskan FC (96 well plates, shaking, 3 filters (405 nm, 450 nm, 620 nm)) 736-0355 Multiskan FC with incubator (96 and 384 well plates, shaking, incubation, 3 filters (405 nm, 450 nm, 620 nm))

736-0356

Large colour screen and visual internal software offer ease of use


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

access to the most commonly used protocols in routine laboratories. In addition, the internal software contains both qualitative and quantitative calculations for single, dual and kinetic measurements offering flexible data handling capabilities. The internal software memory can store up assays protocols and results can be saved to a USB memory stick for easy transfer to a computer.

The internal software is available in English, German, French, Spanish, Russian, Chinese, Japanese and Portuguese.

Thermo Scientific SkanIt Software for optimal computer control

The highly visual and logical user interface of the SkanIt Software, makes instrument control and data handling for both research and routine applications easy. The graphical step list feature allows straightforward setup of any assay. Comprehensive inbuilt calculations, such as: blank subtraction; quantitative curve fit; qualitative classification and kinetic calculations, as well as the versatile reporting tool, make data reduction with SkanIt Software trouble-free. Using the ready-made demo sessions in the SkanIt Software, basic single, dual- and kinetic measurements are performed quickly with only a few mouse clicks. Furthermore, ready-made sessions for many common assays can be downloaded for the SkanIt Software online from the Reading Room (www.thermo.com/readingroom), source for comprehensive information for all Thermo Scientific microplate readers.

What is an antimicrobial treatment?

The handle and the dispensing button of the new Finnpipette F1 are made of antimicrobial polymer. Microbes, such as bacteria, fungi and algae, are found everywhere around us,

and they are also present in the human skin. Normally they are not

harmful, but in some cases they may cause deterioration of the material they grow on. Pipettes are handheld devices and even when the user observes strict cleanliness, microbes from his/her hands may contaminate the pipette. Antimicrobial treatment of the pipette handle parts protects the device from microbial growth on its surface thus preventing deterioration of the polymer parts.

How does it work?

The active ingredient of the antimicrobial material is silver in the form of silver ions. The ions are slowly released from the inorganic matrix via an ion-exchange mechanism. The

release is slow, but fast enough to maintain an effective concentration on the surface of the material. Silver ions are taken up by microbial cells and interrupt critical functions, such as DNA replication, resulting in the death of the microbes. The antimicrobial effect of the material used is long-term and silver inhibits the growth of a broad spectrum of microorganisms.

Testing the efficacy of the antimicrobial treatment

The antimicrobial effect of the material was evaluated according to ASTM standard E2180. The standard describes a test method to evaluate (quantitatively) the antimicrobial effectiveness of agents incorporated or bound into or onto mainly flat hydrophobic or polymeric surfaces. The test organisms used were Escherichia coli, Staphylococcus aureus, Candida albicans and conidiospores of Aspergillus niger.

Contamination of the antimicrobial polymer pieces was carried out by pipetting 0,2 ml of the cell or conidiospore suspension on test pieces that were stored in a horizontal position throughout the experiments.

After complete drying of the suspensions, the amount of colony forming units (cfu, a measure for viable cells) was determined after 4 h and after 24 h.After 4 h, a reduction of cfu was seen for all four test organisms. After 24 h, the reduction was improved for each micro-organism, except in those cases where 100% reduction was already achieved at the 4 h mark, see Figure 1. These results show that the antimicrobial material results in a significant reduction of microorganisms, demonstrating the efficacy of the antimicrobial polymer.

Please note: The antimicrobial treatment does not protect users or others against bacteria, viruses, germs or other disease organisms. Always clean this product thoroughly after each use.

0 20 40 60 80 100

E. coli

S. aureus

C. albicans

A. niger

% reduction

4 h

24 h

Figure 1. Reduction of model microorganisms on an antimicrobial polymer. The colony forming units were determined at 4 and 24 hours after inoculation.

Thermo Scientific Finnpipette® F1 antimicrobial feature

USB port for flexible data transfer

Disclaimer: The data in this technical note is subject to change without prior notice.


GeneMole® is a new bench-top automated system for extraction of nucleic acids. The system processes from 1 to 16 samples run and handles sample volumes ≤ 200 µl. Reagents are supplied in sealed strips (MoleStrips™) according to the “one-strip one-sample” principle.

GeneMole® automated system:

Reproducible extraction of high quality DNA from 1-16 blood samples

Extracting pure DNA from whole blood is challenging. Blood from vertebrates, except birds, has few DNA containing

cells (as little as 1-2%). The large amounts of haemoglobin and plasma proteins can clog up extraction matrices and inhibit downstream applications. Plus there is always a risk that blood samples contain harmful infectious agents.

GeneMole® is a small scale automated system particularly suitable for extracting DNA from whole blood from any vertebrate. The gentle magnetic bead based separation technology extracts DNA

suitable for most downstream applications. Problems like fragmentation of DNA and clogging, which are frequently encountered with spin columns, are avoided. The closed GeneMole® system also reduces exposure to potentially infected blood compared to any manual extraction method.

This report aims to demonstrate GeneMoles® ability to extract high quality DNA from blood from many different sources.

methodsThe GeneMole® instrument and MoleStrips™ DNA Blood (pre-filled reagent strips) were used to isolate DNA from blood drawn from humans and various domestic animals. DNA was extracted from 100 µl blood and then eluted in 200 µl Tris-HCl. Avian blood has nucleated erythrocytes which gives a very large amount of DNA per sample volume. The chicken samples were therefore diluted in PBS prior to extraction (4 µl blood diluted to a final volume of 200 µl). Fresh blood, frozen blood and blood samples stored in different anti-coagulants were tested. Aliquots of pure DNA were used as template for DNA sequencing, pyrosequencing, long-range PCR and quantitative real-time PCR.

Figure 1. GeneMole® instrument with a MoleStrips™ reagent cartridge (enlarged).

Figure 2. DNA extraction with GeneMole® gives highly reproducible results (CV < 5%). This figure shows DNA extracted from 16 parallels of a human blood sample.


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

GeneMole® automated system:

Reproducible extraction of high quality DNA from 1-16 blood samples

Results

The yield and quality of the extracted DNA (measured by NanoDrop or standard spectrophotometer) are listed in table 1. High quality DNA was obtained from all the tested samples.

Automated DNA extraction gave highly reproducible results without any visible crosscontamination (figure 2). The DNA was free from inhibitors and compatible with downstream analysis methods like DNA sequencing (MegaBACE and ABI), multiplexed, long-range- and quantitative real-time PCR (ABI). The DNA quality was also adequate for SNP typing by pyrosequencing (figure 3). For data on other downstream methods contact your local VWR specialist.

ConclusionGeneMole® yields high quality DNA from human and domestic animal blood. Automation eliminates operator-to-operator variations, and the system provides some protection from infected blood samples. GeneMole® is therefore an ideal extraction system for laboratories with need for processing 1-32 samples per day, and it is suitable for genetic testing of humans and animals, veterinary diagnostics, biobanking and genetic research.

Data for domestic animals were kindly provided by Ulla Gustafson and Marie Wibe at Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences.

NB! GeneMole™ is not certified for IVD, and should only be used for research applications

Table 1. Yield and quality of genomic DNA extracted from blood from different species by GeneMole®. The values are averages of 16 samples run in parallel (one donor). Yield depends on white blood cell counts, and will therefore vary from individual to individual

Figure 3. DNA from blood extracted by GeneMole® used for pyrosequencing. The results show three different porcine SNPs associated with meat quality. These genotypes were identified by Professor Leif Andersson’s group at SLU, Uppsala, Sweden and are used as genetic markers in breeding programs around the world.

Species Sample Elution OD Yield Concentration volume (µl) volume (µl) 260/280 (µg) (ng/µl)

Calf 100 200 1,82 4,0 20,0Chicken 4 100 2,02 8,2 82,3Cow 100 200 1,77 3,0 15,2Dog 100 200 1,69 5,6 27,8Horse 100 200 1,86 4,2 21,0Human EDTA 100 200 1,92 4,6 22,8Human Citrate 100 200 1,89 4,4 21,8Human Heparin 100 200 1,80 5,0 25,1Pig 100 200 1,81 4,5 44,9Sheep 100 200 1,79 4,2 20,8

Description Pack Cat. No.GeneMole® instrument 1 731-3051MoleStrips™ DNA Blood (test-kit) 32 731-3050MoleStrips™ DNA Blood 64 731-3053MoleStrips™ DNA Tissue (test-kit) 32 731-3052MoleStrips™ DNA Tissue 64 731-3054


New insights into DNA decontamination

Advanced experiments in gene technology demonstrate that even small amounts of free DNA molecules are sufficient to cause infections, recombination or biological transformations [1,2]. The complete decontamination of equipment and surfaces from any DNA molecules is important for biological containment and safety, as well as for preventing artifacts in PCR amplification experiments. Using new methods to detect extremely low levels of DNA molecules, we investigated the molecular mechanism of action of various commercially available DNA decontamination reagents and found that with high concentrations of DNA and short incubation times none of the conventional reagents destroyed DNA molecules efficiently despite their corrosive or even toxic properties. AppliChem have developed the new and unique decontamination reagent, DNA-ExitusPlusTM, and in this paper it is compared with other conventional products demonstrating it’s fast and efficient destruction of nucleic acids that is gentle on you, your equipment and the environment.

Decontamination reagents use different combinations of three molecular principles for destruction or inactivation of genetic material. Modification and denaturation mask but do not destroy the genetic information encoded in DNA strands and there is a risk that they may be chemically re-activated. Safe DNA decontamination depends on the degradation of DNA into very small fragments. Figures 1 and 2 show the results of sensitive quantification of the fragmentation process by the novel DNA-ExitusPlusTM and conventional agents. No degraded DNA pieces were found when products relied on modification or

denaturation methodology. So based on our current knowledge of gene technology and the principles of recombination, we concluded that these reagents are no longer sufficient. Even reagents that did show degradation of DNA cause only partial destruction and some very large DNA fragments that contain the complete genetic information, still survived treatment (Figure 1).

Only Applichem’s DNA-ExitusPlusTM achieved rapid and efficient degradation with it’s strand breaking activity independent of the size and sequence of the DNA fragments since it works by chemical action and not an enzymatic activity. To verify this efficient degradation of DNA molecules by DNA-ExitusPlusTM, PCR analysis was used (Figure 3) proving that no amplifiable DNA templates are present. Defined DNA samples were dried on the inner surface of reaction tubes, followed by treatment with DNA-ExitusPlusTM. Many different non-standardised PCR tests are used to demonstrate successful DNA decontamination but with large DNA control templates, low DNA concentrations, and high dilutions in the washing steps, evidence is very limited. Only by using PCR analysis in combination with a sensitive DNA degradation test can one be sure that the DNA is only modified or masked and may cause difficulties later on. It is clear that spraying DNA-ExitusPlusTM on lab surfaces ensures complete decontamination.

Another severe disadvantage of conventional reagents is revealed in a test for their corrosive potential. For this purpose different metal plates were incubated with aliquots

Dr. Wolfram H. Marx (AppliChem GmbH, Darmstadt)Dr. Karlheinz Esser (multiBIND biotec GmbH, Dortmund)Prof. Dr. Thomas Lisowsky (multiBIND biotec GmbH, Dortmund)

The first reagent really knocking out DNA contamination: DNA-ExitusPlusTM

Figure 1. DNA degradation by selected conventional DNA decontamination reagents in comparison with DNA-ExitusPlusTM. Sample preparation:- 200 ng CCC plasmid DNA (7kb) in 10 ml

H2O + 5 ml sample - Incubation 3 or 10 min. at 20 °C - Denaturation for 3 min. at 92 °C- 200 ng DNA sample per laneC: Control 200 ng CCC plasmid DNA in 10 ml H2O + 5 ml H2O; M: Marker 1Kb Ladder; X1, X2, X3, X4 = competitor’s products; D = conventional DNA-Exitus; D+ = DNA-ExitusPlusTM.

Figure 2. Degradation of small DNA fragments by DNA-ExitusPlusTM. To test the degradation of small fragments, 500 ng of a 750 bp PCR product and the corresponding primer were treated for the indicated time (1, 2, and 5 minutes) with DNA-ExitusPlusTM. + 5 µl DNA with 5 µl DNA-ExitusPlusTM; C Control 5 µl DNA with 5 µl water; M molecular weight marker 1 kb ladder. After the treatment, the DNA was denatured for 2 minutes at 95 °C.

Figure 3. PCR test for the complete removal of DNA contaminations by DNA-ExitusPlusTM. Test DNA (0,1 to 1 ng) was lyophilised on the inner surface of PCR tubes, incubated for 20 secs with sterile water or DNA-ExitusPlusTM, then washed twice with 100 µl of sterile water. For the PCR test 50 µl of each reaction mixtures were taken containing primers for the amplification of the control and test DNA. 1 ng of control DNA in each sample proves that the PCR reaction is not inhibited so that amplification of a DNA band corresponding to the test DNA indicates that intact DNA molecules are present. If the test DNA is completely degraded the PCR reaction should not amplify any DNA fragment for this template. The negative control with sterile water (H2O) exhibits DNA bands for the test and control templates whilst after treatment with DNA-ExitusPlusTM only the fragment of the control DNA is amplified.



Figure 4. Corrosive potential of selected conventional DNA decontamination reagents in comparison with DNA-ExitusPlusTM. Metal plates representing typical laboratory materials and equipment where treated with 10 ml of each indicated reagent for 20 minutes. 0 = sterile water; X2, X3 = competitor’s products; D = conventional DNA-Exitus; D+ = DNA-ExitusPlusTM. In some cases one observes a polishing effect by the removal of dirt or oxide layers.

Figure 5. Autoclaving of recombinant bacteria leads to partial DNA degradation. Equal volumes of either water (-) or DNA-ExitusPlusTM (+), respectively, where added to 50 ml cultures of recombinant E. coli and autoclaved at 120 °C and 1,2 bar for 20 minutes. After autoclaving 10 ml aliquots of these cultures where loaded onto an analytical agarose gel. Please note the DNA degradation by DNA-ExitusPlusTM (+) resulting in fragments smaller than 20 base pairs.

Figure 6. PCR analysis of the autoclaved E. coli cultures from Fig 2. The recombinant E. coli cultures contained a plasmid bearing the ampicillin resistance gene (AmpR-Gen). Aliquots (2 ml) of the cultures where analysed by PCR for the presence of the complete AmpR gene. (-) = E. coli plus water; (+) E. coli plus DNA-ExitusPlusTM; (K) = E. coli plus DNA-ExitusPlusTM plus 2 ng template for the AmpR gene (positive control to prove that PCR is working); (M) molecular weight marker.

of the products. Figure 4 shows that all conventional products contain aggressive chemicals with corrosive, harmful or even toxic effects including ingredients such as azides, mineralic acids like phosphoric acid or hydrochloric acid, aggressive peroxides or strong alkaline substances like sodium hydroxide. Even after only 20 minutes of incubation irreversible damage of metal surfaces are observed, but DNA-ExitusPlusTM shows no metal corrosion or indeed in other tests (data not shown) no damage to many different plastic surfaces either. DNA-ExitusPlusTM therefore offers a gentle and environmentally safe alternative that degrades and removes all DNA molecules with high efficiency but is also neither toxic nor corrosive.

Finally, autoclaving is believed to be an effective method for DNA decontamination although limited to use with heat-resistant materials and equipment that fit into the equipment. Under the standard autoclaving conditions, DNA molecules are degraded into fragments of 20 to 30 base pairs and PCR analysis demonstrates that even after autoclaving, larger DNA fragments can be identified [1], especially when nucleic acids are protected by protein envelopes (e. g. viruses) or within microorganism (e.g. bacteria). The reaction time of DNA-ExitusPlusTM corresponds to an autoclave run time drying within 10 to 20 minutes after spraying on a surface. Due to its chemical composition, DNA-ExitusPlusTM is not heat-sensitive and does not contain volatile and harmful ingredients. Figures 5 and 6 show the results of comparing it’s effects on bacterial cultures and nucleic acids at elevated temperatures to autoclaves. Only the addition of DNA-ExitusPlusTM leads to an efficient degradation of bacterial DNA, while under the standard conditions (aqueous solutions, medium) the controls are always positive in terms of undegraded / partially degraded DNA.

SummaryIt is clear that performing a PCR test alone to prove the complete removal of DNA

molecules is not sufficient as such a PCR test will be negative for decontamination reagents that modify or mask DNA, whilst failing to remove or destroy it. The true determination of the decontamination potential of a reagent requires PCR analysis in combination with a DNA degradation test. In addition the use of autoclaving has to be re-evaluated, since the latest data show that DNA from viruses and microorganisms are not inactivated properly.

These are the outstanding and unique characteristics of DNA-ExitusPlusTM: I. Catalytic and cooperative effects of

the components cause a very rapid non-enzymatic, non-sequence-specific degradation of DNA and RNA molecules

II. All components of DNA-ExitusPlusTM are readily bio-degradable and not harmful or toxic for humans

III. No aggressive mineralic acids or alkaline substances are used. Equipment and materials are not damaged or corroded even after prolonged incubation times

IV. No harmful aerosols when spraying on surfaces.

Literature:[1] Elhafi, G. et al. (2004) Microwave or autoclave

treatments destroy the infectivity of infectious bronchitis virus and avian pneumovirus but allow detection by reverse transcriptase-polymerase chain reaction. Avian Pathology 33, 3003-306.

[2] Burns, P.A. et al.(1991) Transformation of mouse skin endothelial cells in vivo by direct application of plasmid DNA encoding the human T24 H-ras oncogene. Oncogene 6(11), 1973-1978.


The testing of a new generation tissue homogeniser

Bertin Technologies has designed a new generation homogeniser using bead-beating technology. This system, Precellys-24

(Figure 1 and Figure 2), allows homogenisation of a large range of biological samples from soft to hard and even elastic, simply by varying the bead type and speed. A figure of 8 motion rapidly gyrates the beads to grind from 1 to 24 samples in individual sealed tubes ensuring no cross contamination doubt between the tests. These tubes also make sample storage easy. Handling 24 samples in one sample preparation step means that processes can be very fast with 200 trials being homogenised in just 30 minutes.

Tissue homogenisation is a key process for Pharmacokinetic ADME and Pharmacology study tissue sample analysis using HPLC-

tandem Mass Spectrometer system (LC-MS/MS). Homogenisation simplicity and efficiency; cross contamination, analyte recovery and stability; are important factors that affect the data quality and high throughput (HPT) productivity. The more traditional method of tissue homogenisation involves a mechanical shearing force process through blending or grinding that produces heat, which may cause analyte instability. Some fragile

tissues, such as rat or mouse lymph node and tumours, are also very difficult to handle with mechanic blenders. These tiny pieces must be manually ground, which is a really time consuming.

An ADME study, with rat brain, gonad, heart, kidney, lung and spleen samples were performed

to evaluate the versatility of the Bertin beads beating technology. Including a pharmacological study of tumour and lymph node samples were also used to investigate the reproducibility and analyte recovery of the method.

Experimental Procedure1. Sample PreparationAll tissue samples were weighed out to around 0,5 g directly into the Precellys 2 ml bead tube. To 1 part weight of tissue, 1 or 2 parts weight of buffer (de-ionised water) were added. The sample tubes were then placed on the Presellys-24 for homogenisation based on the following protocols (Table 1). In this experiment, only metallic beads were used. An aliquot of 100 mg homogenate of each sample was weighted out into a 96 well 1 ml cluster tube. Blank matrix homogenate was also weighed out for the preparation of calibration standards (STD), quality control samples (QC) and matrix blanks (BLK) in the sample plate. To each 100 mg homogenate, 400 µl acetonitrile with internal standard (ISTD) was added for analyte extraction. After centrifugation at 3000 rpm for 20 minutes, the supernatant was injected onto a HPLC/Tandem Mass Spectrometer (LC-MS/MS) for sample analysis.

2. HPLC and Mass Spectrometry ConditionsAn Agilent 1100 series HPLC pump was connected with Applied Biosystems Mass Spectrometer API4000 for drug candidate and metabolite analysis. Waters XTerra MS C18 (5 µm, 2,1 x 50 mm) analytical column was used for the chromatography. A 1-minute linear gradient of acetonitrile / 10 mM ammonium acetate was applied for the analyte elution with flow rate of 700 µl min-1. Positive ions were acquired in the multiple reaction monitoring mode (MRM) under APCI ion source. Nebuliser temperature was set at 400 °C.

Results and DiscussionProductivity: The efficiency of tissue homogenisation was significantly improved by 5-20 fold depending on the tissue type. In this experiment, only tumour samples were tested for the productivity. For each tumour sample, at least 1 minute was spent when using manual grinding. But

Bertin Technologies

has designed a

new generation

homogeniser using

bead-beating

technology

Figure 2. Precellys-24Figure 1. Sample tubes with beads



it only took 1 minute to homogenise 24 samples when Precellys-24 was used (Figure 3). There was a 5 minute break time between batches. So only 7 minutes was needed to complete 24-48 samples.

Recovery: To evaluate the analyte recovery, tumour samples were cut into two portions before homogenisation. Three samples from each time point were tested either using manual blending or beads beating. No significant difference was observed between the two methods (Table 2).

Reproducibility: To evaluate the reproducibility, each tumour sample was cut into three portions and prepared in different analytical batches. The process was reproducible within analytical runs (Table 3).

Versatility: In this study, all types of tissues were successfully homogenised and analysed using an LC-MS/MS system. To quantitatively determine the analyte concentration, a nine non-zero level calibration curve at the linear range of

1-5000 ng/ml was prepared for each analytical run monitored by three levels of quality control samples at low, medium and high concentration. To accept the analytical analysis, the measured concentration of the standard at each level must be within ±20% of the nominal concentration. Figure 4 summarises the accuracy of the analytical run for each type of tissues, which demonstrated an excellent accuracy for all tissue sample analyses.

ConclusionPrecellys-24 is a suitable and reliable system for a wide range of small animal tissue or small size of tissue homogenisation. Sample size should be adjusted to be less than 0,5 g for harder tissues, such as tumour, kidney and heart. Precellys-24 is the best homogeniser for handling very fragile tissues, like rat lymph node and tumour samples. It is also the best choice for other tissues weighing around 20-50 mg. When the protocol is set up and validated, sample preparation process remains the same with no bias in analysis.

For more information please contact your VWR Sales Specialist

Sorry - Bertin products are not available in France or Germany

0

10

20

30

40

50

60

70

80

90

100

24 Samples 48 Samples 72 Samples 96 Samples

Blender Precellys-24

Min

utes

-4.00

-3.00

-2.00

-1.00

0.00

1.00

2.00

3.00

4.00

brain gonad heart kidney lung spleen lymphnode

tumor

Figure 3. Productivity comparison: Bertin technology vs. blending methodFigure 4. Average accuracy of analytical run for rat brain, gonad, heart, kidney, lung, lymph node and tumour tissue samples

Ave

rag

e A

ccu

racy

(%

Bia

s)

Table 1. Tissue Homogenisation ProtocolsTissue type Brain Gonad Heart Kidney Lung Spleen Lymph node TumourOriginal weight (g) 1.-1,5 1,5-3 0,7-2 2-2,6 1-1,5 0,5-1 0,02-0,2 0,2-0,5Sample weight (g) ~ 0,5 ~ 0,5 ~ 0,5 ~ 0,5 ~ 0,5 ~ 0,5 0,02-0,2 0,2-0,5Protocol 1 cycle 1 cycle 2 cycles 2 cycles 1 cycle 1 cycle 1 cycle 2 cycles

6500 6500 6500 6500 6500 6500 6500 6500rpm 25s rpm 25s rpm 25s rpm 25s rpm 25s rpm 25s rpm 25s

Table 2. Analyte Recovery Comparison of Rat Tumor Samples Concentration Determined (ng/mL)

Tumour (mean of 3) Time-point 1 Time-point 2 Time-point 3 Time-point 4Precellys-24 17567 22867 5530 559Manual Blending 18407 22933 5737 586

Difference (%) -4,56 -0,29 -3,6 -4,61

Table 3. Reproducibility of Rat Tumour Sample Analysis Concentration Determined (ng/mL)Analysis Batch No. Time-point 1 Time-point 2 Time-point 3 Time-point 4Run 1 17400 24200 4390 560Run 2 17100 24000 5500 496Run 3 18200 20400 6700 620Mean 17567 22867 5530 559S.D. 464 1746 943 50,6CV% 2,64 7,64 17,06 9,06


Microarray technology

has enabled biologists

and medical researchers

to conduct large scale

quantitative experiments.

The most commonly

used method for DNA

hybridisation is static,

where slides are covered

with a glass coverslip

which are then sealed in

a humid container and

hybridisation is achieved

overnight in a water bath

without agitation. However,

such static methods rely

on the diffusion of the

target to the surface-bound

probes, and thus it is a rate

limited process. Mixing of

the hybridisation solution

should minimise localised

target depletion and result

in improved detection and

uniformity of low-abundance

gene transcript signals.

A comparison of static and 3D-rotation methods

The CapitalBio BioMixerTM II Hybridisation Station helps the aqueous hybridisation mixture spread evenly over the microarray

surface by continuous 3D rotation in a temperature controlled environment. This continuous 3D mixing allows each individual sample spot to react uniformly with all of the reagent solution, preventing local depletion of low abundance targets and results in enhanced reaction signal intensity and consistency. It produces demonstrable benefits, with visibly improved quality of the hybridisation signals and better chip-to-chip reproducibility (1-5). These improvements have been attested and documented in independent assessments by the MAQC microarray assessment study undertaken by the FDA (USA) and reported in Nature Biotechnology (1). In combination with CapitalBio spotted array slides and LuxScan 10K-A fluorescence scanner, the BioMixer II hybridisation station produced arguably the highest quality of array images of spotted arrays examined in this comprehensive study.

Here in this report we compared the standard static hybridisation method under a coverslip with the use of the CapitalBio BioMixerTM II, with particular

regard to acrossslide homogeneity and reduction of signal variability.

methodsA human genome 70-mer oligonucleotide microarray (oligonucleotide set version 2.0) manufactured by CapitalBio consists of 5’ amino acid-modified 70-mer probes (synthesised by Operon) representing 21,329 well-characterised genes. For printing, the oligonucleotides were dissolved at 40 mM concentration in CapitalBio DNA Spotting Solution and spotted on amino-modified slides produced by CapitalBio.

DNA in hybridisation solution was denatured at 95 °C for 3 min and added on the spotted slides, then covered with a LifterSlipTM coverslip (Erie Company) and sealed in a CapitalBio hybridisation chamber to obtain the required humidity. After closing the chamber tightly it was either submerged in a water bath and incubated at 42 °C overnight, or placed in the hybridisation compartment of the BioMixer II Hybridisation Station overnight at a rotation speed of 8 rpm and a temperature of 42 °C.

Figure 1. Typical hybridisations resulting from use of the static method (A) and the BioMixerTM II Hybridisation Station (B).

A B

BioMixer II 3D rotation method



After washing and drying, the arrays were scanned with a confocal scanner LuxScanTM 10K and the images obtained were then analysed using LuxScanTM 3.0 software (both from CapitalBio).

ResultsTotal RNAs from two types of cells derived from human peripheral blood mononuclear cell (PBMC) preparations were labelled with Cy5 and Cy3 fluorescent dyes, respectively, and pooled to hybridise on the microarrays. For each slide an overlay picture combining the Cy5 and Cy3 channels was created. Figure 1 shows typical examples of a slide hybridised in the BioMixerTM II Hybridisation Station and a slide processed using the static hybridisation method. These images clearly show that the slides processed in the BioMixerTM II signals are much more homogeneous and have a higher signal-to-noise ratio than the slides processed by normal static methods. To further emphasise the difference in signals between the two processes, data was extracted from the images and compared.

Homogeneity of backgroundTo estimate the homogeneity of hybridisation, we analysed the whole array in three parts: part I (blocks 1 to 16); part II (blocks 17 to 32); part III (blocks 33 to 48). The amount of signal was measured from the spot foreground and the background from a local ring around the spots, respectively. After deriving the value of standard deviation of background, the coefficient of variation (CV) for each part was calculated (see Figure 2). It can be seen clearly that the coefficient of variation of the background is significantly lower in each part of the slide hybridised on the BioMixerTM II Hybridisation Station than on slides hybridised by the static method.

Signal-to-backgroundThe ratio between signal and background (S/B) is much higher for hybridisations processed on

the BioMixerTM II Hybridisation Station compared with those processed by the static method, and on average was some 1,8 times higher for the automated BioMixerTM II Station. This means that a greater dynamic range of signals can be obtained on the BioMixerTM II. More constant signal-to-background ratios are also seen with the BioMixerTM II, whereas the S/B ratios using the static method show higher variation. Thus, significantly better hybridisation reproducibility is obtained by using the BioMixerTM II Hybridisation Station (see Figure 3).

ConclusionIn this study we compared the results of a normal static hybridisation protocol with those of a hybridisation processed on the BioMixerTM II Hybridisation Station. The results consistently showed that there is enhanced signal intensity, lower background and improved signal uniformity of hybridisations resulting from use of the BioMixerTM II Hybridisation Station. These findings indicate that BioMixerTM II Hybridisation Station can be used to assay gene expression over a large dynamic range, and will aid the analysis of low abundance transcripts.

Reference List1. Patterson, TA. et al., Performance comparison of one-

color and two-color platforms within the microarray quality control (MAQC) project. Nature Biotechnology, 2006, 24(9): 1140-1150.

2. Yu, J. et al., Identification of the gene transcription and apoptosis mediated by TGF-beta-Smad2/3-Smad4 signaling. J Cell Physiol, 2007, 215: 422-433.

3. Xia, Q. et al., Microarray-based Gene Expression Profiles in Multiple Tissues of the Domesticated Silkworm, Bombyx mori. Genome Biology, 2007, 8: R162.

4. He, YQ. et al., Comparative and functional genomics reveals genetic diversity and determinants of host specificity among reference strains and a large collection of Chinese isolates of the phytopathogen Xanthomonas campestris pv. Campestris, Genome Biology, 2007, 8: R218.

5. Guo, Y. et al., Distinctive microRNA profiles relating to patient survival in esophageal squamous cell carcinoma, Cancer Research, 2008, 68(1): 1-8.

6. Liu, H. et al., Microarray-based analysis of stress-regulated microRNAs in Arabidopsis thaliana, RNA, 2008, 14: 836-843

Molecular biology

Figure 2. Comparison of the CV background of slides processed on the BioMixerTM II with slides processed by the static method.

Figure 3. Comparison of the Signal-to-Background of BioMixerTM II with that of the static method.

Figure 2. CV Background Figure 3. Signal-to-background ratio


Materials and method

Equipment• Biomek® 2000, 8-channel head (Beckman Coulter)• Kingfisher96(ThermalFisher)• MagneticSeparationDevice(OmegaBio-tek

MSD-01)• 96microplate(AxygenP-96-450-V-C)• 96deep-wellplate(OmegaBio-TekEZ9602)• Tips

Genomic DNA isolation

Genomic DNA isolation from fresh or frozen human blood preserved with different anticoagulants are processed by following stand protocol provide with the Mag-Bind Blood DNA kit. DNA isolation from dried blood from collection paper, saliva and buccal swab is processed with a slightly modified protocol by digest sample in TL Buffer (can be purchased separately) with Proteinase K first. The lysate are then transferred to a new plate and follow the stand Mag-Bind DNA isolation protocol to isolate genomic DNA.

Amplification

PCR reactions were performed using purified DNA as template with primers for human ß-actin gene.

Restriction enzyme digestion

Purified genomic DNA is digested with three common restriction enzymes (EcoR I, Hind III, BamH I) for eight hours. The samples are analysed by agarose gel electrophoresis.

DNA stability analysis

To test the stability of the purified DNA, Isolated genomic DNA from blood was stored at room temperature for 1-3 weeks. The DNA was analysed by gel electrophoresis.

Isolation of genomic DNA from different volumes of human whole blood

Genomic DNA is successfully isolated from 2 µl, 5 µl, 50 µl, 200 µl and 500 µl fresh human blood anticoagulant with EDTA using Mag-Bind Blood DNA kit. 1 µl of Purified DNA from each sample was used as a template for PCR amplification. The

gel electrophoresis results (Figure 1 and Figure 2) demonstrate that highly intact genomic DNA can be purified with Mag-Bind blood DNA kit and the purified DNA are free of contaminants and PCR inhibitors.

Isolation of genomic DNA from human blood samples using different anticoagulants

Genomic DNA can be successfully isolated from 200 µl blood samples with different anticoagulants (EDTA, Sodium Citrate and heparin). The gel electrophoresis results ((Figure 3 and Figure 4) demonstrate that highly intact genomic DNA can be purified from these samples using Mag-Bind blood DNA kit and the purified DNA are free of

Isolation of DNA from Blood samples using conventional methods is increasingly proving to be limited and time consuming factor for research, forensic, veterinary applications. The Mag-Bind Blood DNA system is a magnetic bead-based genomic DNA system that is designed to work on a wide range of samples and other body fluids includes fresh or archived blood samples, saliva, buccal swabs. This method allows rapid and efficient extraction of genomic DNA without the use of centrifugation. The procedure can be fully automated on a robotic liquid handling instrument.

Isolation of DNA from blood samples and body fluids using E-Z 96TM Mag-Bind Blood DNA system

TY, Zhu, Guo,Qi

Figure 1. A: genomic DNA was isolated from 2 µl, 5 µl, 50 µl, 200 µl, 500 µl blood using Mag-Bind Blood DNA Kit. All of purified DNA was loaded on gel when processing 2 µl and 5 µl blood. 15% of purified DNA was loaded on gel when processing 50 µl, 200 µl, 500 µl blood.

M 2 µl 5 µl 50 µl 200 µl 500 µl C M

Figure 2. 10% (2µl, 5µl,) and 1% (50-500 µl) purified DNA was used as template in a 25 µl PCR with primers for human ß-actin gene. The expected PCR product size is 300bp.

1

2



contaminants and PCR inhibitors. The average yield of DNA from 200 µl blood is between 7-9 µg with OD260/280 ratio 1,80-1,90 (Figure 5)

Isolation of genomic DNA from dried blood samples, saliva and buccal swabs

We have also successfully isolated DNA from saliva, dried blood (on collection paper), buccal swab by using Mag-Bind Blood DNA Kit with KingFisher 96 instrument (Figure 6 and Figure 7). The samples were first mixed with TL Buffer (Can be ordered separately) and digested with Proteinase K. The cleared lysate are transferred to a new 96 KingFisher DW plate. DNA is isolated by following protocol modified to fit KingFisher instrument.

Restriction digestion

Library construction, southern blot and other applications require digestion of genomic DNA with restriction enzymes. Figure 8 shows that the genomic DNA isolated from blood sample can be successfully digested three of most common restriction enzymes (EcoR I, Hind III, BamH I).

Stability of purified DNA

Genomic DNA purified using the Mag-Bind Blood DNA Kit was stored at room temperature for 1-3 weeks to evaluate the stability of the isolate DNA. The DNA was analysed by gel electrophoresis. The results show no degradation after 3 weeks of storage in room temperature (Figure 9).

Conclusion

The Mag-Bind Blood DNA kit is a provides a easy and reliable system to isolate PCR-Ready DNA from various biological samples. It can be easily to be adapted into robotic liquid handling instruments for fully hands-free processing.

Molecular biology

Figure 5. The average yield and OD260/280 ratio was analysed by spectrophotometer (DU640, beckman).

Figure 6. Electrophoresis analysis of genomic DNA isolated from saliva and dried blood spot using Mag-Bind Blood DNA kit. 10% of total eluted DNA from each sample was loaded into the gel.

Figure 7. Electrophoresis analysis of genomic DNA isolated from buccal swab using Mag-Bind Blood DNA kit. 10% of total eluted DNA from each sample was loaded into the gel.

Description Preps Cat. No.Mag-Bind blood DNA kit 1 x 96 OMEGM6211-00Mag-Bind blood DNA kit 4 x 96 OMEGM6211-01Mag-Bind blood DNA kit 20 x 96 OMEGM6211-02

Figure 3. Genomic DNA were isolated from 200 µl human blood with different anticoagulant. 10 µl of purified DNA was loaded on gel.

Figure 4. 1 µl of purified DNA was used as template in a 25 µl PCR with human B-actin primers. The expected product size is 300bp.

Figure 8. Restriction digest of genomic DNA purified with E.Z.N.A. MagBind Blood DNA Kit. 0.5 µg genomic DNA was digested with 1U restriction enzyme.

7

9

6

8

5

4

3


Fluorescence technology is a routinely used tool in life science research. Fluorescence reagents provide the advantages of highest sensitivity and specificity paired with low toxicity. Today they are one of the best choices to trace the presence of bio-molecules in cells and other biological systems.

Thermo Scientific Fluorescence Dyes – broadest portfolio and many new alternative dyes

Continuous development in the field of fluorescence detection requires a high range of flexibility for dye modified biopolymers.

Our portfolio (see table 1) provides a wide range for single, double or multiple labeling of oligonucleotides with classic fluorescence dyes or recently developed alternatives.

Our fluorescence modification possibilities allow the application of all common methods, like FISH, In situ Hybridisation, sequencing and genotyping. Fluorescence and Quencher pairs are available for qualitative and quantitative detection of PCR products (qPCR/ Realtime PCR). In addition, Fluorescence modifications can be combined with non-fluorescence modifications like Phosphate, Amino, Thiol, Biotin or could be attached to oligonucleotides with incorporated special bases like LNA, wobbles, RNA and PTO. The flexibility for many combinations allows the adaptation to your special requirement.

A broad range of fluorescence dyes were invented by Dyomics and new methods, techniques and

instruments have recently been developed using these dyes.

In the field of biochip detection Dy-415, Dy-550, and Dy-631 have been tested (1). For sequencing and fragment analysis on LiCor instruments Dy-681, Dy-781 and Dy-800 have been established (2,3,4). Another publication describes the development of a miniaturised exo-nuclease-based FRET assay using Dy-647 and DyQ 660 (5) as a FRET pair.

All Dyomics Dyes are compatible with solid phase chemical synthesis of oligonucleotides. Therefore we are able to extend our portfolio, offering a choice for alternative Dyes. (see table 2)

1) For all dyes please refer to our license information2) Depending on application, adaptation of instruments may

be necessary

References:1. Glasenapp C., Mönch W., Krause H., Zappe H., Biochip

reader with dynamic holographic excitation and hyperspectral fluorescence detection, Journal of Biomedical Optics, 12(2007)014038

2. Rothenstein D., Haible D., Dasgupta I., Dutt N., Patil B.L., Jeske H.,Biodiversity and recombination of cassava-infecting begomoviruses from southern India, Archives of Virology, 151(2006)55-69.

3. de Almeida R.A., Heuser T., Blaschke R., Bartram C.R., Janssen J.W.G., Control of MYEOV Protein Synthesis by Upstream Open Reading Frames, J. Biol. Chem, 281(2006) 695-704.

4. Kaltenpoth M., Strohm E., Gadau J., Polymorphic microsatellite markers for a solitary digger wasp, the European beewolf (Philanthus triangulum; Hymenoptera, Sphecidae), Molecular Ecology Notes 4(2004)589-592.

5. Käppel N.D., Dankbar D.M., Gauglitz G., DNA Quantification in Nanoliter Volumes, Microchim Acta, 154(2006) 65-71.

Advantages• Highsensitivity• Highspecificity• Lowtoxicity• Broadrangeofclassicdyes• Broadrangeofalternativedyes,likeDyomics,NuLight,Bodipy,• Mostpossibilitiesforcombinationswithfluorescencedyes,quenchersor

other modifications• Combinationwithspecialbases,likeLNA,wobbles,RNA,siRNA,PTO• ProbesforqPCR/RealtimePCRassays(Taqman,MolecularBeacons,FRET)• RoutineHPLCpurificationandMScheck

Applications• FISH,InSituHybridisation• qPCR/RealtimePCR• FRET(FluorescenceResonanceEnergy

Transfer)• Sequencing• Genotyping• MicroarrayStudies



Table 2 alternative to2) available

DY - 405 Alexa 405 3’ / 5’

DY - 415 DEAC, Alexa 430 3’ / 5’

DY - 490 Alexa 488 3’ / 5’

DY - 495 FAM 3’ / 5’

DY - 505 Rhodamine 110, Rhodamine Green 3’ / 5’

DY - 549 NED, Alexa 555 3’ / 5’

DY - 590 Texas Red 3’ / 5’

DY - 615 Alexa 610 3’ / 5’

DY - 634 Alexa 633

DY - 649 Alexa 647 3’ / 5’

DY - 681 IRD 700, Cy 5,5 3’ / 5’

DY - 781 IRD 800 3’ / 5’

DY - 800 IRD 800 3’ / 5’

Table 1Fluorescence dye Absorption

maximum [nm]Emission maximum [nm]

Extinction coefficient [M-1 cm-1]

Emission area

Quencher

BHQ

-0

BHQ

-1

BHQ

-2

Eclip

se

Tam

ra

Dab

cyl

Cascade Blue 400 420 28.000

lmax

453

nm

(3

90-5

10 n

m)

AMCA 353 422 19.000Pacific Blue 416 451 36.000

lmax

495

nm

(430

-520

nm

)

Marina Blue 365 460 20.000

lmax

522

nm

(460

-550

nm

)

DY-415 418 467 34.000Bodipy 493/503 493 504 89.000

lmax

534

nm

(480

-580

nm

)

Bodipy FL 504 513 68.000Oregon Green 500 499 519 78.0006-Fam 495 520 83.000

lmax

544

nm

Fluorescein 495 520 83.000DY-495 495 520 70.000ATTO 488 501 523 90.000Oregon Green 488 496 524 70.000Rhodamine Green 505 527 68.000Rhodamine Red 505 527 120.000Oregon Green 514 511 530 70.000DY-505 505 530 80.000Tet 521 536 73.000ATTO 520 516 538 110.000Bodipy 507/545 508 543 69.000Bodipy R6G 528 547 70.000Joe 527 548 71.000Yakima Yellow 531 549 84.000Bodipy 530/550 535 552 62.000

lmax

579

nm

(550

-650

nm

)

ATTO 532 532 553 115.000Hex 535 556 73.000Bodipy 558/568 560 568 74.000Bodipy 564/570 563 569 142.000Cy3 550 570 150.000DY-548 558 572 150.000DY-554 551 572 100.000DY-555 547 572 100.000DY-556 548 573 100.000Bodipy TMR 535 574 50.000DY-547 557 574 150.000NuLight Dy547 557 574 150.000DY-549 560 575 150.000Tamra 544 576 90.000ATTO 550 554 576 120.000DY-560 559 578 120.000Bodipy 576/589 575 588 83.000Bodipy 581/591 581 591 136.000ATTO 565 563 592 120.000Redmond Red 579 595 52.300DY-590 580 599 120.000Rox 576 601 82.000Texas Red 583 603 116.000Cy3.5 588 604 116.000Bodipy TR 588 617 45.000ATTO 590 594 624 120.000DY-610 610 630 80.000Bodipy 630/650 632 640 100.000DY-615 621 641 200.000DY-630 636 657 200.000DY-632 637 657 200.000DY-633 637 657 200.000DY-631 637 658 200.000DY-634 635 658 200.000Bodipy 650/665 651 660 100.000ATTO 647 645 669 120.000Cy5 650 670 250.000DY-635 647 671 200.000DY-636 645 671 200.000NuLight Dy647 653 672 250.000DY-647 653 672 250.000DY-648 653 674 250.000DY-650 653 674 220.000DY-652 654 675 220.000DY-649 655 676 250.000DY-651 656 678 220.000ATTO 655 663 684 125.000Cy5.5 675 694 250.000DY-677 673 694 180.000DY-675 674 699 180.000DY-676 674 699 180.000ATTO 680 680 700 125.000IRDye 700 685 705 170.000DY-681 691 708 140.000DY-680 690 709 140.000DY-682 690 709 140.000ATTO 700 700 719 120.000DY-700 707 730 140.000DY-701 706 731 140.000DY-730 732 758 240.000DY-732 736 759 240.000DY-731 736 760 240.000DY-734 734 766 240.000Cy7 743 767 200.000DY-752 748 772 270.000DY-750 747 776 270.000DY-751 751 779 270.000DY-780 782 800 170.000DY-782 783 800 170.000DY-776 771 801 240.000IRDye 800 787 807 200.000DY-831 844 - 220.000

Fluorescence dye Absorption maximum [nm]

Emission maximum [nm]

Extinction coefficient [M-1 cm-1]

Dyomics Megastoke-Dyes* for multicolour detectionHigh stoke shift allows excitation at simular wavelengths and detection at different wavelengthsDY-485XL 485 560 50.000DY-510XL 509 590 50.000DY-480XL 500 630 50.000DY-481XL 515 650 50.000DY-520XL 520 664 50.000DY-521XL 523 668 50.000

Licences information:- All licenses cover only R&D application of our customer.- BHQ1, BHQ2, BHQ3 are licensed from Biosearch Technologies, Inc.- Cy dyes are a trademark from GE Healthcare- IRDyeTM products are licensed and sold under agreement with LI-COR. IRDyeTM

products must not be sold outside Europe- Molecular Probes’ dyes are licensed from the NIH- Yakima Yellow, Redmond Red and EclipseTMDark Qu. are made and sold under

license from EPOCH Biosciences, Inc.- NuLightTMDYes are sold under the license of Thermo Fisher Scientific- Dyomics Dyes are sold under the license of Dyomics GmbH


Quantitative 16S Ribosomal DNA detection using ABsolute QPCR master mix for diagnosis of central vascular catheter associated bacterial infection.

In many patient populations, central vascular catheters (CVCs) are routinely used for the administration of drugs, fluids or intravenous feeding solutions. CVCs are a major risk factor for bloodstream infections. The majority of hospital-acquired bloodstream infections are associated with the use of a CVC. These infections are associated with considerable costs for patients and health providers.

CVC infection is caused predominantly by bacteria, particularly staphylococci, with a small proportion of infections

being attributable to fungi. Staphylococcus epidermidis is the most common cause of CVC-associated bacteremia. CVC infections caused by S. epidermidis can often be effectively treated with antibiotics infused through or locked in to the CVC. Many of the other bacterial and fungal causes of infection respond poorly to antibiotic therapy alone, and patient outcome is improved by removal of the CVC.

Many CVCs are removed unnecessarily because current diagnostic methods for CVC associated infection are unreliable, particularly in the case of patients exposed to antibiotics. The level of diagnostic uncertainty associated with these methods is such that infection involving the CVC is confirmed for fewer than 25% of the CVCs that are removed because of suspected infection.This application note describes a novel

approach based on the quantitative detection of bacterial DNA in blood samples drawn through the CVC as developed by Simon Warwick et al. 1 Use of this method has the potential to diagnose CVC associated bacterial infection and substantially reduce unnecessary CVC removal, thus contributing to better patient care1.

GoalTo demonstrate that the use of a quantitative PCR assay using primers and probe targeted to bacterial 16S-ribosomal DNA has the potential to diagnose CVC-associated bacterial infection and substantially reduce the unnecessary removal of CVCs.

Experimental conditionsPatient PopulationA population of patients undergoing intravenous feeding were selected for study. These patients were not routinely treated with prophylactic antibiotics (which might compromise culture-based diagnostic methods) and had relatively few risk factors for disseminated bacterial infection other than the CVC. Samples collected from 46 patient episodes (13 patients) were analysed. These 46 patient episodes included 16 episodes of probable bacterial CVC infection and one fungal CVC-associated infection. There were 52 CVC samples in total because in six episodes samples were collected from a second lumen of a double-lumen CVC. There were 28 paired blood samples (where blood was taken through the CVC and also from a peripheral vessel). Blood cultures were not collected from four episodes. For all but three patients, samples were taken when patients presented at the out-patient clinic for review (with or without signs or symptoms compatible with CVC associated infection). Patients with signs or symptoms compatible with CVC associated infection were reviewed within 24 hours whenever possible.

Sample preparation4 ml blood samples were collected into EDTA anticoagulation tubes, directly from the CVC and also, when acceptable to the

patient, from a peripheral vessel by aseptic technique. These samples were sent through the routine sample transport system (at ambient temperature) to the laboratory andstoredinaliquotsat–70˚C.Sampleanalysis was performed by laboratory staff with no knowledge of the patient condition. Samples excluded from this analysis were those that were >72 hours old on arrival in the laboratory, those collected within 14 days before or after the day of onset of an episode of probable CVC infection (those collected on the day of onset were included), and those collected while the patient was on intravenous antibiotic therapy.

Method developmentA total of 119 16S ribosomal DNA (rDNA) sequences comprising 56 bacterial species were retrieved from Gen Bank and aligned by using BioEdit (Ibis Biosciences), and highly conserved regions were identified. Possible primers and probes were selected and then validated by using the Primer Express software provided by Applied Biosystems. The ability of these primers and probes to detect 10 pg of purified DNA from 30 bacterial species was determined, and those described by Nadkarni et al.2 were selected for use in the study.

DNAwasextractedfrom200μlaliquotsof EDTA-anticoagulated whole blood. The samplewasmixedwith1.200μloffreshlyprepared 0,17 M ammonium chloride and incubated at room temperature for 30 minutes. Following centrifugation at 11.600 x g for 10 minutes, the pellet was washedtwicewith500μlofsterilesaline(0,9% wt/vol). By using the QIAamp DNA minikit (Qiagen), the pellet was re-suspended in180μlofQiagenATLbuffer(containingEDTA and sodium dodecyl sulphate) and exposed to six freeze-thaw cycles (cycling between–70˚Cand50˚C),withvortexingbetween, before being heated in a boiling water bath for 10 minutes. The remainder of the extraction procedure was performed according to the manufacturer’s protocol. DNAwaselutedin50μlofbufferfollowinga5 minute incubation. Extracts were stored at –20˚Cbeforeanalysis.

Simon Warwick - Department of Microbiology, St. Bartholomew’s Hospital and the London NHS Trust.



A number of controls were routinely run with each batch of tests. These included blood samples from a healthy individual with and without spiking with bacteria. An extraction control of blood spiked with 10 colony forming units of S. epidermidis/μlwasfoundto yield DNA levels close to the lower limit of detection. Bacterial DNA controls of known amounts (100 pg to 100 fg) and a negative control (with template DNA omitted to detect reagent contamination) were also included in each run.

PCR conditionsReal-time PCRs were carried out by using the ABsolute QPCR Master Mix with ROX in optical 384 well plates. Ultrafiltration of reagents, as suggested by Yang et al.3, was not performed. Reactions mixtures contained (1X) ABsolute QPCR Master Mix, 300 nM concentration each of the forward and reverse primers, a 100 nM concentration of thefluorescentprobe,2μloftemplateDNAandwatertogiveafinalvolumeof20μl.Thecyclingconditionswere95˚Cfor15minutesandthen40cyclesof95˚Cfor15secondsand60˚Cforoneminute.The threshold cycle (Ct) value, which is inversely proportional to the log of the amount of target DNA initially present, was calculated by using SDS software version 2,0 (Applied Biosystems). All samples were run in triplicate.

Statistical methodsThe median Ct value from each triplicate was used in the analysis. The data was modelled in order to estimate detection rates, false-positive rates and potential reductions in unnecessary removals of CVCs. The area under the curve and its 95% confidence interval were calculated by using Roctab in Stata4.

Results and discussionThe diagnosis of CVC-associated infection is often difficult because of dependence on quantitative microbial culture, which is compromised by use of antibiotics, invasive sampling or removal of the CVC. Microbial culture methods may take several days to give a result, and invasive sampling methods may result in embolisation or bacteremia.The advantages of an approach based on quantitative detection of bacterial DNA

drawn through the CVC are that it can work even when patients have been treated with antibiotics and can be automated.The principle of this approach is that the concentration of bacteria (and therefore bacterial DNA) is high in blood drawn through a colonized CVC. The method used in this evaluation would be unlikely to reliably detect the small numbers of bacteria in the blood of patients with bacteremia. The test is rapid, and the cost per test is low once the appropriate equipment is available.

The results suggest that, in this population of patients, a single sample from the CVC is as valuable in excluding the diagnosis of CVC bacterial infection as the comparison of the levels of bacterial DNA in the central and peripheral blood.

In this study there were only a small number of samples from the multiple lumens of a colonized CVC. We found variation in the amount of bacterial DNA in samples taken from the different lumens, which suggests that this test approach might be useful in differentiating which CVC is colonized in patients with multiple CVCs.Although we deliberately excluded samples from patients who had received antibiotic therapy within the preceding two weeks, DNA from bacteria that have been killed by antibiotics can still be detected by PCR in the blood, suggesting that the technique may be useful in patients populations in which antibiotic treatment is more common.

ConclusionThe target for the quantitative PCR is a region of the 16S rRNA gene which is highly conserved in most bacteria. This ensures that the test using ABsolute QPCR Master Mix with ROX has the potential to detect virtually all of the bacterial causes of CVC infection.

References1. Warwick, S. et al. (2004) Use of Quantitative 16S

Ribsomal DNA Detection for Diagnosis of Central Vascular Catheter-Associated Bacterial Infection. J. Clin. Microbiol. 42(4):1402-1408

2. Nadkarni, M. et al. (2002) Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set. Microbiol. 148:257-266

3. Yang, S. et al. (2002) Quantitative multiprobe PCR assay for simultaneous detection and identification to species level of bacterial pathogens. J. Clin. Microbiol. 40:3449-3454

4. Stata Corporation (2001) Stata statistical software, release 7.0. Stata Corporation, College Station, Tex.

Molecular biology

Figure 2. Ct values in patients with probable CVC-associated bacterial infection and patients in whom CVC-associated infection was unlikely. Ct results are medians of three results.

Figure 3. Differences in median Ct values between CVC and peripheral blood sample. (Ct value for CVC blood - Ct value for peripheral blood).

Figure 4. Receiver - operating curve plotting true-positive rate (sensitivity) against false-positive rate (1-specificity), showing the actual data (dashed line) and the modelled receiver-operating curve (solid line).

Figure 1. Relationship between the apparent amounts of S. epidermis and E. coli DNA, and the median Ct with three different sets of 16S rDNA primers and probes. The lowest Ct values indicate the highest concentration of bacterial DNA.


It´s time for 5 PRIME´s - PerfectPure RNA purification kits

High yields of pure, stable RNAThe 5 PRIME PerfectPure RNA Purification kits employ a novel silica membrane column using innovative non-chaotropic chemistries for isolating RNA from a variety of starting materials in as little as 20 minutes. PerfectPure RNA kits eliminate the most common problems in RNA purification, such as overloading and clogging, leading to higher processing capacity and higher yields than competing brands.

The PerfectPure kits are used to purify up to 600μgpersampleofpure,stableRNA,whichisessentially free of genomic DNA, protein, and other

enzymatic inhibitors. No subsequent re-purification is necessary – the RNA is ready for downstream analysis.

Fast and efficient process Total RNA is purified with the PerfectPure RNA purification kit by adding a detergent/salt solution to the sample to disrupt cells and eliminate endogenous RNase activity. When purifying from whole blood, red blood cells are first lysed and white blood cells are collected before addition of the detergent/salt solution.

Cell membranes are disrupted by homogenisation to release the RNA into the lysing solution. Tissue and large-volume white blood cell lysates are then passed through a 5 PRIME Preclear™ column to remove any undesirable debris remaining after homogenization. Lysates are applied to the purification column to bind the RNA and wash away proteins, DNA, and other contaminants. Residual DNA is removed with on-column DNase treatment. Finally, the purified RNA is eluted with DEPC-treated water.

The PerfectPure RNA kits come complete with all the required reagents, pre-mixed and ready to use right out of the box. They are available in a single spin column or 96-well plate format. All necessary disposable components (columns, tubes, plates) are included.

Novel column-based technology eliminates the most common difficulties of RNA purification, including overloading and clogging.

â Stable, high-quality RNA, free of genomic DNA, proteins, and other enzymatic inhibitors

â Fast and efficient process

â Complete kit, ready-to-use out of the box

â Environmental friendly chemistry

The PerfectPure kits can be used to isolate RNA from different starting material like e.g.:

• Wholeblood

• Buffycoat

• Culturedcells

• Tissue

• Fibroustissue

Total RNA from up to 10 ml whole blood

1 Ladder2 - 4 0,5 ml blood5 - 7 3,0 ml blood

8 Ladder9 - 11 4,0 ml blood12 - 13 10,0 ml blood

Total RNA isolated from 10 ml whole blood using the PerfectPure RNA Blood Kit.



It´s time for 5 PRIME´s - PerfectPure RNA purification kits

Molecular biology

Product specifications and performanceRNA binding capacity of column Greater than 600 µgMinimum elution volume 50 µl A260/A280 ratio1 1,8–2,2 RNA size distribution2 Greater than 150 bases

1 Sample diluted in TE buffer.

2 Although PerfectPure RNA is most efficient for isolating RNA species greater than 150 bases in length, in vitro transcribed RNA less than 150 bases in length can be successfully purified. Highly structured RNA molecules that are less than 150 bases in length, such as tRNAs, are selectively excluded from the purification.

Ordering information

Purified RNA is used successfully in the following downstream applications:

• RT-PCRand real-time PCR

• cDNAsynthesis

• Microarray

• Northernhybridisation

• Ribonucleaseprotection assays

• In vitro transcription

RNA Purficiation from

Kit Sample Size Preps Cat. No.

Whole blood and buffy coat

PerfectPure RNA Blood Kit

0,05 – 3,0 ml 10 733-10720,05 – 3,0 ml 50 733-10733,0 – 10,0 ml 50 733-1074

Culture cells Standard throughput

PerfectPure RNA Cell Kit

10 000 – 5 x 107 cells

10 733-107550 733-1076250 733-1077250 (Base Kit) 733-1773

High Throughput

Centrifugation PerfectPure RNA 96 Cell Kit

5x 106 cells/well 192 733-1083768 733-1085

PerfectPure RNA 96 Cell CS Kit

5x 106 cells/well 192 733-1084768 733-1086

Vacuum PerfectPure RNA 96 CellVac Kit

768 733-1087

Tissue PerfectPure RNA Tissue Kit

0,5 – 40 mg 10 733-107850 733-1079250 733-1080

Fibrous Tissue PerfectPure RNA Fibrous Tissue Kit

0,5 – 40 mg 10 733-108150 733-1082

Total RNA from up to 10 ml whole blood

The Agilent Bioanalyzer profile demonstrates the quality and integrity of PerfectPure purified RNA subjected to accelerated stability testing at 4°C for seven days prior to the analysis.


In Vivo Retinal Electroporation using BTX products

phiC31 Integrase confers genomic integration and long-term transgene expression in rat retina

Purpose. Gene therapy has shown promise in animal models of retinal disease, with the most success achieved to date with viral vectors used for gene delivery. Viral vectors, however, have side effects and limitations and are difficult to manufacture.The present study was conducted in an attempt to develop a novel system for long-term gene transfer in rat retinal pigment epithelium (RPE), by using nonviral transfection methods for gene transfer and the integrase from the bacteriophage C31 to confer long-term gene expression by means of genomic integration.

Methods. Efficient nonviral delivery of plasmid DNA to rat RPE in vivo was achieved by using subretinal injection of plasmid DNA, followed by in situ electroporation. Gene delivery was evaluated by analysing enhanced green fluorescent protein (eGFP) expression in frozen sections. In subsequent experiments, a plasmid expressing luciferase, with or without a plasmid encoding the C31 integrase, was delivered to rat RPE. Luciferase expression was followed over time by using in vivo luciferase imaging.

Results. Subretinal injection followed by electroporation yielded abundant transgene expression in the rat RPE. Expression was

strongest 48 hours after delivery. In the absence of C31 integrase, transgene expression declined to near-background levels within 3 to 4 weeks after treatment. By contrast, coinjection of the integrase plasmid led to long-term stable transgene expression throughout the 4,5 month test period. Eyes injected with C31 integrase showed 85-fold higher long-term transgene expression in the retina than eyes without integrase.

Conclusions. Subretinal injection of DNA followed by electroporation affords abundant transfer of plasmid DNA in rat RPE. C31 integrase confers robust long term transgene expression by mediating genomic integration of the transgene. Thesefindings suggest that C31 integrase may be a simple and effective tool for nonviral long term gene transfer in the eye.

Molecular biology

Reference;(1) Chalberg, T., Genise, H,, Vollrath, D., Calos, M., 2005.

phiC31 integrase confers genomic integration and long-term transgene expression in rat retina. Invest Ophthalmos. Cis. Sci. Jun; 46 (6):2140-6

ECM® 830* in vivo electroporation protocol

Tissue preparation:Perform subretinal injections in the superior hemisphere of the animal. Soak BTX Tweezertrodes in PBS and apply to each cornea with negative electrode on the injected eye with 14 mm between electrodes. (Figure 2)

Electroporation settings:Voltage: 140VPulse length: 100 msecNumber of pulses: 5Interval: 950 msField strength: 100 V/cm


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

Tangential flow filtration for small sample volumes as low as 2 ml with the KrosFlo® Research II TFF System

When using tangential flow techniques, most of the process fluid flows along the membrane surface rather than passing

through the membrane structure. Fluid is pumped at a relatively high velocity parallel to the membrane surface. Except for water treatment applications, only a small percentage of the tangential flow along the membrane surface ends up as permeate. In most cell and particle separations, for example, only 1% to 5% of the inlet flow to the membrane device becomes permeate. The remaining 95% to 99% exits the membrane device as “retentate”.

The retentate is recirculated back to the process reservoir and the module inlet such that another 1% to 5% can be removed as permeate. This recirculation process continues in rapid succession generating a significant and continuous permeation rate. See the illustration.Various tangential flow membrane geometries include stacked plate and spiral devices which utilize flat sheet membranes, tubular devices, and shell and tube devices which use hollow fiber membranes. In the case of tangential flow separations, the driving force is the transmembrane pressure (TMP), the difference between the average of the module feed and retentate pressures and the permeate pressure:

PTMP = (Pfeed + Pretentate) / 2 – Ppermeate

Filtrate flow results in a build up of retained components on the membrane inner lumen surface. Generally these components are carried down the length of the hollow fiber and out the end of the module by the sweeping action of the recirculating

Tangential Flow Filtration (TFF) is an efficient way to separate streams that would become quickly plugged if processed by dead-ended techniques.

How Spectrums’ filter module works

Tangential Flow Filtration


fluid. However, under certain conditions a cake layer accumulates on the surface of the membrane. This boundary layer is composed of solids and/or solute macromolecules which are retained by the membrane during the course of filtration. This phenomenon, often erroneously referred to as “concentration polarisation” can affect module performance by reducing the apparent size of the membrane pore. In other words, the cake layer becomes the membrane barrier, a “dynamic membrane”.

The extent of caking is influenced by such fluid variables as the degree of solvation, concentration and nature of the solids and solutes, fluid temperature and operating variables such as solution velocity along the membrane and transmembrane pressure (TMP). Controlling this phenomenon is the key to maximising flux and solute passage and optimizing the process parameters.

Caking can usually be controlled by ensuring adequate fluid velocity at the liquid-membrane wall interface. Fluid velocity is controlled by the pumping rate. Recirculation rates depend on the quantity of fibers in a module and shear rate considerations. Typically, a shear rate of 12 000 s-1 is used for filtration applications and up to 4 000 s-1 is used for perfusion applications. These rates are guide lines only and should be optimised for each process application. Certain applications may work well at reduced rates while others may require rates that are significantly higher. When

protein passage through the membrane structure is important, particular attention should be paid to feed (or recirculation rate). In general, high feed rates allow more efficient protein passage. Depending on the characteristics of the retained components (cells, cell debris, diagnostic particles, etc.), a caking layer can form on the membrane wall

that is actually tighter than the membrane pores. In these instances, high recirculation rate and low transmembrane pressure often help. Variables such as viscosity or shear sensitivity of the solution components may prevent the user from generating enough velocity to reduce membrane caking. Often in these cases, applying a determined amount of back pressure on the permeate helps to prevent caking. Cell perfusion applications frequently use permeate back pressure to prevent caking.The KrosFlo® Research II Tangential Flow Filtration

KrosFlo® digital pressure monitor

KrosFlo® data collection software

FEED RETENTATE

PERMEATE

Cut-a-way view of a single fiber cross flow filtration module



(TFF) System is the ideal system for research and development studies for microfiltration and ultrafiltration applications. The system consists of the KrosFlo® Research II Pump and Pump Head, the KrosFlo® Digital Pressure Monitor, and a disposable flow path, all of which have features that ensure efficient and reproducible TFF processes. The upgraded KrosFlo® Digital Pressure Monitor has both audible low and high pressure alarms for the inlet and permeate and, when combined with the KrosFlo® Research II Pump Drive, has a high pressure pump shutoff that helps maintain membrane integrity and achieve high product recovery. The Pressure Monitor also comes standard with KF Comm, a software program that automatically downloads and graphs the run data including inlet, retentate and transmembrane pressures and flow rates, into an MS Excel® spreadsheet. The disposable flow path eliminates the possibility of cross contamination and allows samples to be concentrated down to as low as 2 mls. Other standard features include: a digital

readout of the flow rate on the KrosFlo® Research II Pump, the new easy-to-use KrosFlo® Research II Pump Head, and adjustable holders attached to the pump drive that secure the process vessels, Spectrum’s HF modules and the entire flow-path.

The KrosFlo® Research II TFF System offers many conveniences over traditional cross-flow membrane systems. The hollow fiber (HF) membrane modules and cross-flow filtration system provide faster and gentler separation that helps avoid membrane fouling and maximises product recovery.The disposability of the modules eliminates not only the potential for cross contamination and costs associated with cleaning and rinsing, but also the difficulties associated with validating re-use membranes.Used in conjunction with HF membrane modules, the KrosFlo® Research II TFF System offers the following advantages: faster process times, superior filtration dynamics, module disposability, lower costs and direct and easy scale-up production volumes.

Sample preparation

Ordering information KrosFlo® Research II TFF Systems:Flow-path type Pump capacity Tubing F/P capacity Cat. No.MiniKros® 2,3 LPM, 220 V L/S 14, 1/16” ID 130 ml/min 515-0207MidiKros® 2,3 LPM, 220 V L/S 16, 1/8” ID 480 ml/min 515-0206MicroKros® Sampler Plus EU Plug 2,3 LPM, 220 V L/S 17, 1/4” ID 1,7 ltr/min 515-0209MicroKros® Sampler Plus UK Plug 2,3 LPM, 220 V L/S 17, 1/4” ID 1,7 ltr/min 515-0210MicroKros® Sampler Plus CH Plug 2,3 LPM, 220 V L/S 17, 1/4” ID 1,7 ltr/min 515-0208

KrosFlo® Research II pump

Spectrum developed about 200 disposable tangential flow filtration modules for the KrosFlo® Research II TFF System: Mixed Ester (ME)-, Polysulphone (PS)-, and Polyethersulphone (PES) fiber, in three fiber ID’s and in 8 different pore sizes. Please ask for a full list of filter modules from your local VWR sales office.


Pall Life Sciences AcroPrep filter plates for efficient sample preparation

The AcroPrep 96-well plate configuration contains a proprietary design that creates an integral seal with either single or multiple

membrane layers. For example, the placement of a porous prefilter over a more restrictive micro-porous membrane combines the benefits of fast flow with the ability to more completely clarify particulate laden solutions.

Beyond high throughput clarification, the AcroPrep 96 UF plate configuration can be used to rapidly concentrate and recover biomolecules in either

vacuum or centrifugal modes using safety sealed ultrafiltration membranes. High performance UF membranes with well-defined pore structures have a long and successful history of separating molecules according to size.

Use of ultrafiltration for protein and nucleic acid sample preparation

Biomolecule purification involves a complex series of steps where molecules are selectively separated using size or a variety of other biochemical properties. Many steps require desalting and buffer exchange as well as sample concentration to prepare the biomolecule sample for the next step in purification. Desalting/buffer exchange can be a critical step in the process where yields and biological activity can be significantly reduced. It can be accomplished using methods such as gel filtration, size exclusion HPLC, dialysis, and ultrafiltration (UF). Compared to the use of non-membrane based processes, filtration with UF has a number of technical advantages:

• Simultaneousprocessing– desalting and sample concentration occur in the same step

• Speedandsimplicity– ultrafiltration is fast and easy to perform

• Biologicalactivity– no organic extraction is required and the ionic and pH milieu is maintained

• Versatility– useful at low and high throughput modes, with or without automation, and at varying temperatures

Historically, most filtration devices were designed to process individual samples. Pall Life Sciences introduces a family of novel microfiltration and ultrafiltration devices which are capable of processing 96 samples simultaneously. AcroPrep 96 filter plates were designed to be low biomolecule binding, low-weeping, chemically-resistant, and compatible with the guidelines established for automated plate handling systems.



Using UF to separate molecules based on size has proven to be one of the most effective ways to process biological samples. Although the primary basis for separation is molecular size, other factors such as molecule shape and charge also play a role. Molecules larger than the membrane pores are retained on the surface of the membrane, while smaller molecules pass through the membrane into the filtrate in this very efficient process.

The efficiency of AcroPrep 96 UF filter plates to remove salts and other small molecules is not at the expense of sample recovery. Figure 1 demonstrates that using AcroPrep 96 10K, recovery of ovalbumin proteins (45 kDa) at two different concentrations, 0,1 and 1,0 mg/ml, were greater than 90% with salt removal efficiencies of also greater than 95%. Using BSA (66 kDa) and a 30K MWCO membrane, we observed similar protein recoveries and desalting effeciencies.

The AcroPrep 96 filter plates containing UF membranes provide a fast and simple means to efficiently desalt, buffer exchange and/or concentrate samples. The versatile multi-well format allows one to process up to 96 samples

simultaneously, or as little as one sample at a time. AcroPrep 96 plates are equally effective with centrifugal or vacuum filtration.

Multi-well, membrane-bottom plates for high throughput sample preparation and detection procedures

• Reducecrosstalk– proprietary sealing technology individually seals membrane in each well, reducing well-to-well crosstalk

• Addversatility– available in 96 and 384 well formats. Available in a variety of membranes and configurations, plate colours, well volumes, and outlet tip lengths for use in a multitude of sample preparation and detection processes

• Assureconsistency– designed in accordance with the standards of the Society for Biomolecular Screening (SBS). Single-piece design strengthens these plates for automated applications

• Gainconfidence– the chemically resistant/biologically inert polypropylene housing construction is low protein and nucleic acid binding, and durable when used with harsh chemicals

• Makeitconvenient– serialised barcode allows for use in automated tracking systems

For technical and product information about multi-well plates, visit www.pall.com/lab.

To order Pall Life Sciences products, contact your VWR Representative or visit www.vwr.com.

Sample prepatation


The Novagen® WideScreen™ Group has launched the first wave of unique assay panels for the bead-based Luminex xMAP Technology platform. The focus has been on creating assays that detect the phosphorylation status of key proteins in disease-related signalling pathways. These assays will enable researchers to determine the effect of candidate compounds on multiple pathways, to allow a better understanding of on and off target effects.

WideScreenTM Multiplex assays for the Luminex® xMAP® Technology platform

WideScreen™ EpiTag™ ERK pathway assays

•UtilisingadvancedEpiTag™Technology

•Quantitatebothtotalandphosphorylatedproteinsfrom the same sample, in the same well

•Simplesamplepreparation-onelysisbufferforallproteins

WideScreen™ EpiTag™ assays utilise EpiTag™ Technology licensed from Epitome Biosystems Inc., which detects protein and phosphoprotein fragments rather than the traditional detection of intact proteins. This approach provides a high degree of specificity and the ability to multiplex assays for a protein’s total and phosphorylated levels without cross reactivity. In addition, the assay can accurately measure concentrations of phosphorylated target proteins as synthetic phosphopeptides are used to generate standard curves.


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

WideScreen™ RTK assays

•MostextensiveRTKbead-basedassaypanel

•Multiplexanalysissavestime,sampleandmoney

•Moredata-lesstime!

The WideScreen™ RTK Assays consist of a series of phosphotyrosine-specific RTK assays and companion total RTK assays. The phosphotyrosine assays utilise specific capture antibodies and a broad-spectrum phosphotyrosine detection antibody. The total RTK assays, which include standards, allow the signals from the phosphotyrosine assays to be compared to the amount of RTK in the sample.

EpiTag™ is a trademark of Epitome Biosystems, Inc. Luminex® and xMAP® are registered trademarks of Luminex Corporation.

For further information about WideScreen™ Multiplex assays please request a brochure from your local VWR sales office.


During the last 25 years, several gel-based techniques have been developed to rapidly and economically reveal single-base mutations in DNA fragments. C.B.S. Scientific has developed 3 different instruments that can be used to scan mutations by DGGE, CDGE, TTGE or SSCP. Choose the instrument that fits your application from the chart.

3 Different instruments for Mutation Screening

Both DGGE and TTGE rely on establishing a gradient of either solvent (urea/formamide) or temperature in which the target fragments

will undergo conformational transition (melt). This sequence dependent information will determine the theoretical melting behavior of the target fragment after PCR amplification. If the sequence is known, then primer/probe design can be made using certain software design programs (1). If not, the melting range can be revealed by running perpendicular DGGE/TTGE gels. The third mutation detection technique, SSCP, differentiates between normal and mutant duplex DNA by denaturing the DNA to form single stranded molecules of equal length. These molecules can reanneal onto themselves and based on the varying degree of intrastrand base pairing, form different three dimensional structures. These structures differ in electrophoretic mobility and can be separated on a non-denaturing polyacrylamide gel.

DGGE and CDGEDenaturing Gradient Gel Electrophoresis (DGGE) is a powerful genetic analysis technique that can be used for detecting single base changes and polymorphisms in genomic (2,3), cloned, and PCR amplified DNA (3,4). Two of the most valuable uses for DGGE in human genetics are in directly detecting single base changes that cause disease and in detecting polymorphisms with DNA probes for genetic-linkage analysis. In DGGE, conformational transitions of multiple nucleic acid complexes are induced by an increasing concentration of solvent (urea/formamide) at a constant temperature. Clinical applications of DGGE include a rapid and effective method for screening samples for genetic mutations and variants. Also, DNA fragment melting points can be determined using perpendicular DGGE (2). In contrast to DGGE, CDGE (Constant Denaturant Gel Electrophoresis) uses a single solvent percentage to induce partial

APPLICATIONS

• DetectingsinglebaseDNAchanges/mutations

• DetectingPolymorphismsindoublestrandedDNA

• DeterminingDNAfragmentmeltingpoints

• DetectsSingleStrandConformationalPolymorphisms

Instrument DGGE TTGE SSCP CDGEDGGEK-2401 √ √TTGEK-2401 √ √ √DTSK-2401 √ √ √ √

Figure 1. DGGEK-2401 with power supply on the left.

Figure 2. The TTGEK-2401 and DTSK-2401 system look the same on the exterior as shown on the right. The DTSK-2401 has an interior coil for cooling capability.



melting of DNA fragments as they enter the gel. The disadvantage of CDGE is that only a single melting domain can be interrogated.

TTGEDestabilisation of nucleic acid complexes can be studied using acrylamide gels that contain a uniform solvent concentration as in CDGE, but with an increasing temperature gradient (6). Since the temperature of the entire gel is uniformly raised over a period of time, this technique has been termed ‘TTGE’, or Temporal Temperature Gradient Electrophoresis (7). This technique incorporates many improvements over DGGE/CDGE especially when studying multiple melting domains (8).

SSCPSingle Strand Conformational Polymorphism (SSCP) reveals differences in electrophoretic mobility between normal and mutant single strands of DNA (9). In SSCP, normal and mutant duplex DNA are denatured to form single stranded molecules of equal length. These molecules can re-anneal onto themselves and based on the varying degree of intrastrand base pairing, form different three dimensional structures. These structures, differ in electrophoretic mobility and can be separated on a chilled non-denaturing polyacrylamide gel.

The electrophoretic mobility of these “conformers” change depending on temperature and buffer ionic strength. For accurate characterisation of mutations within these re-folded single strands, it is essential that the buffer temperature be tightly controlled within each electrophoresis procedure, usually between 4-15 ºC. If the gel temperature is not precisely controlled, the resolution will suffer because of the loss of intrastrand base pairing and change in the overall shape of the 3-dimensional conformer.

The DTSK instrument is equipped with a large diameter cooling coil in the anode buffer chamber. The cooling coil is connected to an external

refrigerated water bath (chiller). The DTSK automatically mixes the buffer and regulates the overall temperature; it is capable of holding the buffer temperature within 0,05 ºC of the set temperature assuring consistent intra- and inter-gel conditions. The DTSK System is the only system listed below that can perform all three applications, DGGE, TTGE and SSCP.

References:1) Tian-Jian Chen, Richard G. Boles, Lee-Jun Wong, Detection

of Mitochondrial DNA Mutations by Temporal Temperature Gradient Gel Electrophoresis. (1999). Clinical Chemistry 45:8, 1162-1167

2) S.G. Fischer and L.S. Lerman, (1983) PNAS 80:15793) Guldberg, P., Henriksen, K.F., and Guttler, F. (1993).

“Molecular analysis of phenylketonia in Denmark: 99% of the mutations detected by denaturing gradient gel electrophoresis.” Genomics 17:141-146

4) Moyret, C., Theillet, C., Puig, P.L., Moles, J-P., Thomas, G. and Hamelin, R. (1994). “Relative efficiency of denaturing gradient gel electrophoresis and single strand conformation polymorphism in the detection of mutations in exons 5 to 8 of the p53 gene.” Oncogene 9:1739-1743.

5) Sheffield, V.C., Beck, J.S., Kwitek, A.E., Sandstrom, D.W., and Stone, E.M. (1993). “The sensitivity of single-strand conformation polymorphism analysis for the detection of single base substitutions.” Genomics 16: 325-332

6) K. Yoshino, K. Nichigaki, Y Husimi. (1991) Temperature sweep gel electrophoresis: a simple method to detect point mutations. Nucleic Acids Res. 19:31-53

7) J. Bjorheim, S. Lystad, A. Lindblom, U. Kressner et al. (1998) Mutation analysis of KRAS exon 1 comparing three different techniques: temporal temperature gradient electrophoresis, constant denaturant capillary electrophoresis and allele specific polymerase chain reaction. (1998) Mutat Res 403: 103-112

8) A.H Fanrleitner, N. Kreuzinger, G.G Kavka et al (2000) Comparative analysis of denaturing gradient gel electrophoresis and temporal temperature gradient gel electrophoresis in separating Escherichia Coli uidA amplicons differing in single base substitutions. Letters in Applied Microbiology 30: 427-431

9) Orita M., Sekiya T., and Hayashi K,. 1990. DNA sequence polymorphisms in Alu repeats. Genomics 8: 271-278

10) Orita M., Sekiya T., and Hayashi K,. 1989a. Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5: 874-879

11) Orita M., Iwahana H., Kanazawa H., Hayashi K, and Sekiya T., 1989b. Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc. Natl. Acad. Sci. 86: 2766-2770

Electrophoresis

Description EU Plug UK Plug CH plugDTSK-2401-220 DTS Electrophoresis System Bundle (DGGE, CDGE, TTGE & SSCP) with programmable temperature ramping heater/stirrer and cooling capability (4-Place), 220 volt, CE

730-1311 730-1312 730-1313

TTGEK-2401-220 TTGE Electrophoresis System Bundle (DGGE, CDGE & TTGE) with programmable temperature ramping heater/stirrer (4-Place), 220 volt, CE

730-1318 730-1319 730-1320

DGGEK-2401-220 DGGE Electrophoresis System Bundle for DGGE & CDGE, with non-programmable, non-ramping heater/stirrer (4-Place), 220 volt, CE

730-1324 730-1325 730-1326

All systems feature:

•2dualcassettes

•Tankwithsafetyinterlock lid

•Mini-peristalticpump

•Gradientmaker

•8combs

•8setsofspacers

•4setsofglassplates

•8GelWrap™gaskets

•Whitespringclamps

•Buffersiphonpump

Your European Distribution Partner

AustriaVWR International GmbHGraumanngasse 71150 WienTel.: 01 97 002 0Fax: 01 97 002 600E-mail: [email protected]

BelgiumVWR International bvba/sprlHaasrode Researchpark Zone 3Geldenaaksebaan 4643001 LeuvenTel.: 016 385 011Fax: 016 385 385E-mail:[email protected]

DenmarkVWR - Bie & BerntsenTransformervej 82730 HerlevTel.: 43 86 87 88Fax: 43 86 87 90E-mail: [email protected]

FinlandVWR International OyPihatörmä 1 C 1FI - 02240 EspooTel.: 09 80 45 51Fax: 09 80 45 52 00E-mail: [email protected]

FranceVWR International S.A.S.Le Périgares – Bâtiment B201, rue Carnot94126 Fontenay-sous-Bois cedexTel.: 0 825 02 30 30 (0,15 EUR TTC/min)Fax: 0 825 02 30 35 (0,15 EUR TTC/min)E-mail: [email protected]

GermanyVWR International GmbHHilpertstrasse 20aD - 64295 DarmstadtTel.: 0180 570 20 00*Fax: 0180 570 22 22*E-mail: [email protected]*14 Cent/Minute aus d. dt. Festnetz

IrelandVWR International LtdOrion Business CampusNorthwest Business ParkBallycoolinDublin 15Tel.: 01 88 22 222Fax: 01 88 22 333e-mail [email protected]

Northern IrelandVWR International LtdA10 Harbour Court, 7 Heron RdSydenham Business ParkBelfast BT3 9HBTel.: 028 9058 5800Fax: 028 9080 7812Email: [email protected]

ItalyVWR International s.r.l.Via Stephenson 9420157 Milano (MI)Tel.: 02 332 03 11Fax: 800 152 999E-mail: [email protected]

The NetherlandsVWR International B.V.Postbus 81981005 AD AmsterdamTel.: 020 4808 400Fax: 020 4808 480E-mail: [email protected]

NorwayVWR International ASKakkelovnskroken 1P.B. 45, Kalbakken0901 OsloTel.: 02290Fax: 815 00 940 E-mail: [email protected]

PortugalVWR International - Material de Laboratório, LdaEdifício NeoparkAv. Tomás Ribeiro, 43- 3 D2790-221 CarnaxideTel.: 21 3600 770Fax: 21 3600 798/9E-mail: [email protected]

SpainVWR International Eurolab S.L.Ronda Can Fatjó, nº 11 Edifici Tecnopark, 3 Parc Tecnològic del Vallés 08290 Cerdanyola del Vallés(Barcelona)Tel.: 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 16/188953 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]

Order your copy from your local VWR sales organisation or email at [email protected]

Presenting ...

“All you need for Electrophoresis”This handy brochure is packed with all the products you could need for this indispensable technique. From acrylamide to precast gels for protein or DNA/RNA electrophoresis, to agarose, reagents, buffers, dyes, stains and of course equipment, you will find everything you need to run great gels from this single handbook!

issue 21 autumn 2008 -...

Documents