vwruk.vwr-cmd.com/ex/downloads/life_science/biomarke/0909/VWR_bioMa… · reproducible experimental data . ABgene® SuperPlate™ Genomics Figure 2. Competitor plate (left) may warp

Issue 23Autumn 2009

vwr.com

New VWRbioMarke partnersBiochrom, Biotium, ZyGEM

Great NEW products includingPrecellys 24-Dual Tissue Homogeniser

Novel applicationsBD PureCoat Cultureware,Gold standard PCR plates fromAbgene and new assays from Caymanfor protein or DNA methylation

GenomicsABgene – ABgene® SuperPlate™ - the gold standard in PCR and QPCR plastics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Bertin Technologies– New Precellys® 24-Dual tissue homogeniser and cell lyser . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4Mole Genetics – Benefit from implementing GeneMole® automated DNA and RNA extraction . . . . . . . . . . . . . . . . . . . . . . . 6Omega Bio-Tek – Rapid isolation of high quality genomic DNA from whole blood using the E-Z 96® blood DNA kit . . . 85 PRIME – Genomic DNA from valuable samples – 5 PRIME ArchivePure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10ZyGEM – Rapid reliable DNA prep for mouse genotyping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12BDH/Prolabo – BDH Prolabo Electran® agarose range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Biotium – Environmentally safe and ultra-sensitive nucleic acid gel stains for replacing EtBr . . . . . . . . . . . . . . . . . . . . . . . . . 16

Cell BiologyThermo Scientific – An easy determination of the antioxidant power of beverage samples with Thermo Scientific Multiskan FC Microplate Photometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18Corning – Large scale suspension cell growth and secreted product yields using the Corning® HYPERFlask™ Cell culture vessel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20Sartorius – SuperSpinner D1000 – disposable bioreactor for efficient cell cultivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22Brand – Comparison of adsorption capacity between BRAND immunoGrade™ microplates and different competitors 24BD – PureCoat Cultureware, where life thrives on the surface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26Biochrom – The culture of the cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28Wheaton – Wheaton E-Z ExTraction glass microTubes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30Applichem – Antibody stabilisation on ELISA plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32Cayman – New assays quantify protein or DNA methylation with Cayman Methyltransferase and 5-methyl-2-deoxy Cytidine Assay Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34SoluLink – How to immobilise and produce the strongest signals without background noise . . . . . . . . . . . . . . . . . . . . . . . . 36Spectrum – MediaKap® and MediaKap® PLUS hollow fibre media filters - the smartest way of media filtration . . . . . . . 38BTX – Persistence of cooperatively stabilised signalling clusters drives T-Cell activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

ProteomicsGE Healthcare – Convenient and efficient methods for cell lysis and protein extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42G Biosciences – Rapid fractionation & enrichment of proteins for reproducible protein analysis, including 2D electrophoresis . . . 44Thermo Biopolymers – Milestones towards proteomic standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Supporting Products Pall Life Sciences – Centrifugal Devices - Facilitate pure product with > 90% recoveries in just minutes . . . . . . . . . . . . . 48 Thermo Scientific – Dispense liquids containing proteins more reliably with reverse pipetting . . . . . . . . . . . . . . . . . . . . . . 50

Issue 23 S e p t e m b e r 2 0 0 92

This edition of VWRbioMarke is our biggest journal yet!

Hope you had a great summer and enjoy being back at work with this Autumn edition of VWRbioMarke. Joining us in this bumper issue are Biochrom, Biotium and Zygem – all highly specialist reagent companies offering unique products in the cell biology and genomics arenas.

Our long standing members have plenty to contribute too and the articles are as diverse as ever! Celebrating 100 years of reagent excellence we have a focus on the BDH Prolabo Agarose range that has gone from strength to strength over recent months. As well as innovations from Sartorius, Cayman and Applichem to mention a few!

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. Make sure that you use your budget to best advantage with these special offers from VWR!

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 for the Life sciences microsite and also to register for the Life sciences e-newsletter.

editorial

table of contents

EditorVWR International Europe bvbaResearchpark Haasrode 2020Geldenaaksebaan 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 .

Run77 600 copiesPublication date: September 2009

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

nn 5 PRIME

nn ABgene

nn Applichem

nn BD Biosciences

nn BDH Prolabo

nn Bertin Technologies

nn Brand

nn BTX Harvard Apparatus

nn Cayman Chemical

nn C.B.S. Scientific

nn Corning

nn G-Biosciences

nn GE Healthcare

nn Mole Genetics

nn Nalgene

nn Nunc

nn Omega Bio-Tek

nn Pall Life Sciences

nn Quanta Biosciences

nn Sartorius Stedim Biotech

nn SoluLinK

nn Spectrum Laboratories

nn Thermo Biopolymers

nn Thermo Scientific

nn VWR Collection

nn Wheaton Science Products

VWR I n t e r n a t i o n a l VWRbioMarke Issue 23 S e p t e m b e r 2 0 0 9 3

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

Plate distortion can cause many problems, from plates sticking to thermal cycler blocks post cycling, to crashing automated systems due to the plate falling out of specification . With four times the rigidity of a standard polypropylene PCR plates and enhanced thermal resistance, the ABgene®SuperPlate™ can help solve issues .

Superior engineeringThe ABgene® SuperPlate™ range has been specifically engineered with enhanced thermal resistance to reduce warping that occurs when a plate is exposed to rapid increases and decreases in temperature under thermal cycling conditions . Through a combination of innovative design and specially selected polymer, we have created the most rigid polypropylene plate available on the market .

Testing of the ABgene® SuperPlate™ range has shown that they are four times more rigid than standard polypropylene plates (see Figure 1) . Through the enhanced thermal properties that the ABgene® SuperPlate™ exhibits, warping is now virtually eliminated, reducing robotic handling problems seen with traditional polypropylene PCR plates .

VersatilityABgene® SuperPlates™ are available in three 96 well formats, offering compatibility with most PCR

and QPCR thermal cyclers on the market and are also available in white for QPCR users who require improved sensitivity . All ABgene® SuperPlates™ can be supplied bar-coded to aid the process of sample tacking .

Increased convenienceTo make our range of ABgene® SuperPlates™ even easier to work with, we have added the feature of raised black grid referencing on the plates . As the grid-referenced characters are now readily identified, this helps with plate orientation in manual and automated systems . In addition, black lettering offers quick and easy location of samples within the plate .

All ABgene® SuperPlates™ are designed with raised rims around each well to increase sealing effectiveness . Whether using heat or adhesive seals the raised rim gives an optimal surface around each well for the seal to, this virtually eliminates evaporation and delivers more reliable and reproducible experimental data .

ABgene® SuperPlate™

Genomics

Figure 2. Competitor plate (left) may warp after standard thermal cycling conditions. ABgene® SuperPlates™ (right) shows significant reduction in warping after standard thermal cycling conditions

Figure 1. Plate rigidity testing performed using a tensometer to determine stress required to deflect the plate skirt by 2 and 5 mm.

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

52

Deflection Distance (mm)

Stre

ss/ k

Pa

Standard Plate

ABgene® SuperPlate™

Warping of amplification plates can be a major concern for researches performing PCR or QPCR in both manual and automated platforms.

VWR I n t e r n a t i o n a l VWRbioMarke Issue 23 S e p t e m b e r 2 0 0 94

Precellys®24-Dual tissue homogeniser and cell

lyser is designed for researchers involved in RNA/DNA preparation, proteins preparation,

microorganisms dissociation, and ADME/toxicology research .

Precellys®24 tissue homogenisers are dedicated to the grinding, lysis and homogenisation of biological samples prior to DNA, RNA and proteins extraction . Since 2005, it has defined the new efficiency and quality standard of bead beating . There are now 1000 users worldwide in R&D or routine labs that daily perform such high-throughput sample preparations .

The Precellys®24-Dual enables efficient bead-beating samples preparation in 2 or 7 ml tubes in less than 30 seconds within a temperature controlled environment . Precellys®24-Dual can be operated in multiple configurations in the same build-in tube holder: 0,5, 2 or 7 ml, with or without Cryolys cooling option to suit any sample preparation applications .

A figure-8 multidirectional motion gives shaking energy to the beads that grind-homogenise-lyse the sample thoroughly and efficiently . Cold air is sprayed beside the tubes for cooling samples during homogenisation with patented Cryolys cooling option, keeping temperature sensitive molecules in native state (RNA, Proteins or compound) . When the protocol is set up and validated, sample preparation process remains the same, with no cross-contamination and no bias in analysis .

Precellys®24-Dual is the new evolution of Precellys®24 for preparation of samples in 7 ml vials . Researchers often face the question of the sampling rate they need to obtain representative and reliable results:

• In pharmacology and ADME, irregular repartition of compounds results in variation in toxicological analysis . A 7 ml tube enables whole organs homogenisation (brain, lung, liver) and provides more representative and reliable results (Improved CV%)

• In plant & soil research, detection limits and sensitivity can be improved by increasing the sampling quantity . Seeds and plant sample of up to 5 g can be easily loaded .

Evaluation of the Precellys® 24-Dual in pharmacology study

Precellys®24-Dual has been evaluated in a Drug Metabolism and Pharmacokinetics (DMPK) Department part of the Pre-Clinical Division of Chiesi Farmaceutici (Italy) .

Sample preparation & Analysis 8 rats were intra-tracheally administered with an active drug for respiratory diseases . The animals were sacrificed 15 minutes after administration and the lungs withdrawn . Four whole lungs were homogenised with an Ultraturrax TP18/10 and the remaining whole lungs were homogenised with Precellys®24-Dual . The lungs were homogenised with 3 times their volume of acetonitrile / 0,9% NaCl mixture (50/50 v/v) . Lungs weights ranged from 1,2 g to 1,4 g .

The appropriate lysing kit 7 ml vial with ceramic beads (Cat . No . 432-3759) was used at 6500 rpm for 2 cycles of 20 s with 20 s pause to grind samples with Precellys®24-Dual .

All homogenised samples were processed with acetonitrile (3:1 v/v) for Protein Precipitation and after vortex mixing 10 second, were filtered by MINI UNIPREP (Whatman) syringeless filter devices .10 μl of supernatant were injected in LC/MS/MS system .

Results & DiscussionResults of extraction analyte are given in Table 1 . The analyte levels in the sample are almost the same, so no difference involving the two homogenising techniques are detectable (See Figure 1) .

New Precellys® 24-Dual tissue homogeniser and cell lyserFast, cooling adapter, 2 and 7 ml vials bead-beater for

automated sample preparation

Rat # ULTRATURRAX (Area Ratio)

Rat # Precellys®24 Dual (Area

Ratio)1 8,084 2 13,549

3 7,263 4 5,695

5 6,211 6 6,435

7 7,685 8 7,877

N 4 4

Mean 7,311 8,389

SD 0,806 3,557

CV% 11,029 42,404



The homogenised samples obtained with Precellys®24-Dual were good, apart from a few residual cartilaginous fragments (probably bronchus/trachea). These fragments were also present, but to a lesser extent, in the samples processed with Ultraturrax. The results show that there is no impact on the analyte recovery from matrix.

Reproducibility is good in both cases, taking into account the individual variability. The Ultraturrax, had in general a longer processing time - depending on the samples with results at least 5 times faster using Precellys®24-Dual. Plus with 6 slots for 7 ml tubes, throughput can reach 72 samples/hour without the risk of cross contamination. The Ultraturax requires fastidious and time consuming cleaning steps to

avoid potential contamination between samples.

During processing by both methods, the temperature within the sample after processing could reach 70 ˚C depending on the sample and protocols. Considering the short time of exposure to this temperature (samples were preserved in ice before and after processing), analyte degradation may be not important but the Cryolys cooling option provides extra protection for the samples.

Overall the Precellys®24-Dual is found to be an excellent system for grinding whole organs with a fast high throughput process that’s highly reproducible without cross contamination and with comparable analyte levels extracted.

Genomics

Figure 1

The Precellys range of products:

• Precellys®24 is a high-throughput tissue homogeniser for soft to hard tissues that operates simultaneously up to 24 x 2 ml tubes containing up to 500 mg of tissue or 1,5 ml of cell culture.

• Precellys®24-Dual is designed to prepare samples not only from soft to hard tissues but also from 1 mg to 5000 mg.

• The Cryolys is a patented cooling option that keeps temperature at 4 °C during homogenisation. It allows extraction of stable RNA and native-state proteins. Cryolys is compatible with Precellys®24 and Precellys®24-Dual.

• 0,5 ml, 2 ml and 7 ml lysing kits are impact-resistant tubes prefilled with beads designed for Precellys®24 and Precellys®24-Dual. Our protocol database on www.precellys.com offer the full list of link between samples and lysing kits.

Precellys®24-dual was

successfully evaluated during

beta testing in early 2009

by such market leaders as

Hoffman LaRoche, LundBeck,

Agriculture Canada, Biotec.


GeneMole® does the whole DNA and RNA extraction job for you!

This easy to learn and simple to use system is completely automated; You just apply the blood, cultured cells or tissue lysate on GeneMole® and start the instrument . A short time after purified nucleic acids are collected . Lengthy set-up procedures or partially manual steps required by alternative products are avoided . After all, what is the point of automation if you have to do half the job anyway?

Results independent of the userLaboratories are multiuser environments . Two or more technicians often share the extraction job . Students come and go . Every individual has their own "touch" and techniques, but even small variations can have significant impact on the end results . The GeneMole® instrument provides a consistency which is difficult to obtain from one person to another . It is your reliable assistant for constant DNA and RNA extraction quality .

Simplify when possible!

MoleStrips™ is your insurance for top quality DNA and RNA . GeneMole® uses a proprietary reagent system where buffers are pre-filled in individually sealed cartridges; one MoleStrips™ per sample . The single strip replaces the many bottles and tubes you’re used to: Avoid contaminated reagents, no risk for mixing up of reagents and no chance of mislabelling tubes .

Tailor-made

for all creatures

GeneMole® has been documented for a large range of samples and applications . The Mole Recipes Book gives you an idea of the great variety of DNA and RNA extractions our instrument can help out with . As the number of extraction possibilities is growing all the time, chances are that GeneMole® already covers your specific needs or will do so soon . For the latest update on GeneMole documentation, check www .molegenetics .com/cookbook

Benefit from implementing GeneMole®automated DNA and RNA extraction

Better results faster. Publishing, reimbursements and research financing is all about producing high quality results in a timely fashion. GeneMole® helps you achieve better results faster!

The GeneThe GeneMoleMole®® Recipes Book Recipes GeneGeneMoleMole®® is getting into more and more labs. It is amazing what our is getting into more and more labs. It is amazing what our customers extract DNA and RNA from. We are collecting our own experiences customers extract DNA and RNA from. We are collecting our own experiences as well as our customer’s experiences in a web based recipes book. You can as well as our customer’s experiences in a web based recipes book. You can find it at find it at """#$%&'(%%)*%%)#(%$

Find your favorite organism/application

READ other users experiences, ADD your advices, and SHARE your protocols on:

www.molecookbook.com


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.comGenomics


Rapid isolation of high quality genomic DNA from whole blood using the E-Z 96® Blood DNA Kit

Omega Bio-Tek’s E-Z 96®

Blood DNA Kit provides a fast and safe method for the isolation of genomic DNA from whole human blood. The kit can be used with a wide range of whole blood volumes in a 96 well format and has also been used to isolate viral DNA from body fluids. High quality, double-stranded DNA can be isolated in less than 45 minutes.

The isolation of high quality, double-stranded, high molecular weight genomic DNA from whole blood is a fundamental step in genetic analysis . The increasing usage of genetic applications in clinical laboratories processing large quantities of samples calls for rapid and efficient DNA isolation methods .

The E-Z 96® Blood DNA Kit allows purification of high quality genomic DNA in less than 45 minutes . The isolation protocol was designed for use with fresh, frozen or anticoagulated blood samples . Adjusting the volume of whole blood with water allows DNA purification of sample volumes ranging from 1 μl to 250 µl of whole blood.

Purifications of equal quality and yield were achieved from whole blood samples collected in EDTA-, heparin- or citrate-coated tubes . The E-Z 96®

Blood DNA Isolation Kit can also be used to isolate DNA from other samples such as cultured cells and viral DNA from cell free body fluids (data not show in this report) .

Procedure for Isolation of genomic DNA from whole human blood

The E-Z 96® Blood DNA isolation kit uses HiBind®

membrane technology to bind and purify DNA . The E-Z 96® Blood DNA Kit uses a Lyse-Bind-Wash-Elute procedure . The steps involved in the isolation of DNA from whole human blood and the purpose of each step are listed in Table 1 . The entire procedure takes approximately 45 minutes .

Result

The E-Z 96® Blood Isolation Kit was used to isolate total genomic DNA from 200 μl of whole blood collected in either EDTA-, heparin- or citrate-coated tubes . The purified DNA was analysed for DNA yield and fragment length, as well as compatibility with PCR amplification .

Reproducibility

The reproducibility of the purification process was demonstrated by the isolation of DNA from the same whole blood specimen. DNA yield from 200 µl EDTA-treated blood sample is 6,1 µg ± 0,9 µg (Table 2 and Figure A and B) . The concentration is 25-35 µg/µl.

No

A26

0

A28

0

A23

0

A26

0/28

0

A26

0/23

0

Conc

. (µg

/µl)

Yiel

d ( µ

g)

1 0,1125 0,0618 0,0721 1,82 1,56 28,1 5,6

2 0,1235 0,0718 0,0686 1,72 1,80 30,9 6

3 0,0975 0,0542 0,0513 1,80 1,90 24,4 4,5

4 0,1225 0,0696 0,0557 1,76 2,20 30,6 6,1

5 0,134 0,0740 0,0583 1,81 2,30 33,5 6,5

6 0,1164 0,0650 0,0579 1,79 2,01 29,1 5,8

7 0,1234 0,0701 0,0791 1,76 1,56 30,9 6,3

91 0,1237 0,0680 0,0741 1,82 1,67 30,9 6,1

92 0,1289 0,0712 0,0689 1,81 1,87 32,2 6,3

93 0,125 0,0694 0,0683 1,80 1,83 31,3 5,7

94 0,1301 0,0723 0,0651 1,80 2,00 32,5 6,8

95 0,1215 0,0675 0,0579 1,80 2,10 30,4 6,2

96 0,1139 0,0629 0,0575 1,81 1,98 28,5 5,7

Protocol Step Propose

Transfer the blood sample in 96 well deep well plate Prepare blood for Proteinase treatment .

Add BL buffer and incubate 10-15 minutes at 60 °C. Lyse blood cells and release DNA .

Add ethanol into the sample Adjust binding condition .

Load the sample into the E-Z 96® DNA Plate .Centrifuge for 5 minutes

Bind the DNA to the membrane

Add HB Buffer and centrifuge . Remove the contaminants and inhibitors

Add DNA Wash Buffer and centrifugeFurther removal of contaminants and inhibitors .

Centrifuge the empty plate Dry the membrane

Add Elution Buffer and incubate for 5 minutes .Centrifuge to collect the DNA into a new 96 well plate

Elute the purified genomic DNA

Table 1: The procedure of E-Z 96® Blood DNA Kit

Table 2: The UV Date by Beckman Coulter’s DU-640 Spectrophotometer, Dilution factor is 5. Elution volume: 200 µl.

Figure B

Figure C

Figure D Figure E

9

Innovations in nucleic acid isolation

Figure A

Table 2 and Figure A/B: Genomic DNA was isolated from 96 200 µl human blood samples with EDTA-anticoagulant using the E-Z 96® Blood DNA Kit. OD readings and yields were measured on Beckman Coulter’s DU-640 Spectrophotometer.

Figure C: Genomic DNA was isolated from 96 of 200 µl human blood with EDTA-anticoagulant Using this kit.10 µl of purified DNA was loaded on 0,8% agarose gel.

Figure E: Agarose gel analysis of PCR amplification 2 µl of purified DNA from EDTA, TCA, Heparin anticoagulant blood, as indicated in the picture, was used as a template with primers for the ß-actin gene.


The integrity of genomic DNA isolated using E-Z 96® Blood Isolation Kit was analysed by gel electrophoresis . Figure D illustrates that the majority of the DNA ranged from 20-70 kb when isolated from blood collected in each of the three types of coated collection tubes .

PCR Compatibility:

Compatibility of the isolated DNA with PCR amplification was demonstrated with the eight DNA samples isolated from blood collected in each of the three types of coated collection tubes . The human ß-Actin gene can be successfully amplified from all DNA samples (Figure E) .

Summary:The E-Z 96® Blood DNA Kit is a convenient and reliable DNA purification system that can be used for whole blood samples stored in various anticoagulants . This kit produces thorough yields of high quality DNA . The purified DNA is suitable for PCR amplification .

Product Size Cat. No.E-Z 96® Blood DNA Kit 1 x 96 OMEGD1192-00E-Z 96® Blood DNA Kit 4 x 96 OMEGD1192-01 E-Z 96® Blood DNA Kit 20 x 96 OMEGD1192-02Vac-03 Vacuum Manifold 1 OMEGVAC-03

Genomics

Figure D: Genomic DNA was isolated from 200 µl of human blood using different anticoagulants.10 µl of purified DNA was loaded on 0,8% agarose gel.


Genomic DNA from valuable samples – 5 PRIME ArchivePure

The 5 PRIME ArchivePure DNA purification system is designed to isolate highly pure, high molecular weight genomic DNA from a wide variety of samples. The ArchivePure system employs a gentle non-toxic liquid phase DNA method, which is readily scalable and can be easily modified to perform optimally with a wide range of sample types and sample conditions. This makes the ArchivePure DNA Kits the perfect choice for valuable or unique samples like patient blood and demanding downstream applications such as long term storage or long range PCR.

Today a scientist can choose form a wide range of methods, technologies and kits to purify to genomic DNA . The choice will be made depending on their needs for throughput, convenience, reliability, quality, sample type and cost . For samples that are valuable, scarce or difficult to come by e .g . historical patient blood samples there is often only one chance to isolate the DNA . In such cases reliability of the method, high yields and high purity of the resulting DNA might be considered as the most important factors . The ArchivePure System is ideally suited to meet these demands . It provides the user with simple yet flexible protocols, high quality reagents and reliably results in high purity DNA with a molecular weight of 100 – 20 kb .

Fast and simple protocolThe standard ArchivePure protocol for genomic DNA extraction from blood only requires five steps as depicted in figure 1 . It is completely liquid phase, using only gentle, non-toxic chemicals .

1 . The first step is designed to eliminate the enucleated red blood cells using the highly efficient ArchivePure RBC Lysis Solution (also available separately) .

2 . After pelleting, white blood cells are lysed using the Cell Lysis solution . In this solution samples are stable for up to two years at room temperature .

3 . Optionally the sample can be treated with RNase prior to protein precipitation .

4 . DNA is then precipitated from the supernatant using isopropanol .

5 . Pelleted DNA is solved in a proprietary DNA Hydration solution . In this form the DNA can be archived for long periods with no loss in performance .

This basic protocol can be scaled for sample volumes from 150 µl blood to more than 10 ml.

High quality DNADNA isolated by the ArchivePure process is of the highest quality and purity . This is demonstrated by high A260/A280 ratios of 1,7 – 2 high molecular weight (100 – 20 kb) and proven long term stability . Purified DNA is frequently stored prior to analysis procedures such as DNA amplification or Southern

blotting . Several studies have suggested that the stability of the purified DNA varies depending upon the method used for DNA purification and the storage conditions . For DNA purified from blood with ArchivePure storage data for 14 years are available . As shown in Figure 2 Agarose gel electrophoresis analysis of all purified DNA samples showed that the integrity and quality of the DNA was excellent . None of the DNA samples

ArchivePure DNA Purification Procedure


1

14

Figure 2. Stability of DNAEven after 14 years of storage the ArchivePure purified DNA shows high quality and no obvious signs of degradation. 1) Analysis of 10 replicate genomic DNA samples at 1 year time point (lanes 3-12 samples, 1,14 uncut Lambda DNA, 2, 13 Lambda DAN x Hind III) 14) genomic DNA at 14 years time point

Figure 3. PCR after 14 yearsPurified DNA was evaluated for amplification performance using primers specific to a 1,5 kb target in one of the cytochrome P450 genes (CYP2D6 locus).Lanes 1,11 100 bp Ladder, Lanes 2-10 PCR product from 14 year old DNA

Figure 4. Effect of different anticoagulantsHuman whole blood was collected in tubes containing ACD, EDTA or heparin. DNA was purified in triplicate from blood collected in each anticoagulant following a standard ArchivePure protocol for 300 μl whole blood. The DNA was hydrated in 100 μl of DNA Hydration Solution. To test the effect of each anticoagulant on PCR, DNA was amplified using primers specific to a target site within the Factor V gene. Amplification was not affected when DNA was purified from whole blood samples collected in tubes containing ACD (lanes 2-4), EDTA (5-7) or heparin (8-10) using the ArchivePure DNA Purification Kit.

See our offer in


examined at any of the post-purification time points showed evidence of detectable degraded DNA . Furthermore, the size of all DNA was found to be greater than 50 kb in length when compared with the undigested Lambda DNA size marker (figures A, B) . In addition, the expected 1,5 kb amplicon was amplified for all samples, indicating that the DNA continues to remain of high quality over an extended period of time (figure 3) . If DNA samples are to be archived, we recommend freezing the DNA in aliquots at -20 ºC or -80 ºC .

Feature SpecificationSample Volumes 150 µl – 10 ml whole blood or

bone marrow in common anti-coagulants

Preparation time 25 – 60 minYield range 16 – 50 µg/mlYield average 35 µg/mlSize distribution 95% >50 kb

Table 1. Specifications

Difficult samplesAn additional strength of the ArchivePure System is the fact that the base protocol can be easily adapted to the needs of special sample types . The ArchivePure Blood Kit is suitable for fresh or frozen• Whole blood• Bone marrow• Packed cells• Buffy coat

The ArchivePure procedure is suitable for use with whole blood and bone marrow treated with EDTA, citrate (ACD), or heparin . Ideally, whole blood or bone marrow is collected in EDTA to reduce DNA degradation . However, DNA has been extracted successfully from blood stabilised with other anticoagulants as shown in the figure 4 .

Specialised protocols are available e .g . for blood stains, blood smears, clotted blood, umbilical cord blood or compromised blood samples . Compromised samples include those that have been stored at room temperature for more than 24 hours, at 4 °C for more than 5 days, or samples stored at -20 °C.

Many of the components are also available separately to facilitate such protocols e .g . the RBC Lysis solution . RBC Lysis solution can also be used

stand alone e .g . to deplete bone marrow samples of red blood cells prior to cell isolation .

Downstream applicationsThe high quality of the DNA makes it suitable for any type of downstream applications . As 95% of the purified DNA is longer than 50 kb (typically 100 – 200 kb) ArchivePure DNA is the perfect template for long range PCR reaction (e .g . with the 5 PRIME PCR Extender Kit) or real-time PCR assays . It is equally suited for library construction, southern blotting or genotyping applications such as STR analysis, comparative genome hybridisation or SNP array karyotyping1) .

ConclusionThe 5 PRIME ArchivePure Blood Kit offers an ideal solution for DNA purification from valuable samples . It reliably provides long high quality genomic DNA . The simple and fast basic protocol can easily be modified to provide optimal results with difficult samples .

The high quality and length of the resulting DNA makes it the perfect template for even the most demanding downstream applications and long term archiving .

References1) Dunbar, A.J. et al., Cancer Res. 2008 (68, 12) p. 10349


Tissue rapid reliable DNA prep for mouse genotyping

Mt Erebus, Antarctica… the source of prepGEM® / forensicGEM®

The sex of a mouse can be determined by a PCR method developed by Clapcote and Roder (2005) . This method relies on a differential in the intron sizes of two homologous X and Y linked genes, Jarid1c and Jarid1d . Both loci can be amplified using a single primer pair to generate amplicons of 331 and 302 bp . Sex is ascertained either by qPCR and melt curve analysis, or by PCR and gel electrophoresis .

Small animal genotyping with prepGEMBiomedical research frequently uses mice and rats as animal models . In many cases, a precise genotype of the animals is necessary but often this step is time-consuming . With the number of animals required to generate supported data, reagent and labour costs can become prohibitive and so efficient, low-cost DNA extraction is needed . However, such a method should still be capable of providing template suitable for a broad range of analytical techniques .

Here we describe the benefits of using a DNA extraction kit from ZyGEM Corporation that allows tens or hundreds of samples to be processed in an hour while still producing DNA of a suitable quality for analysis . The prepGEM® kits are designed to be automated on any liquid handling workstation that can carry PCR or peltier plates, or semi-automated using a simple workstation and a thermal cycler . The prepGEM® kit uses less plasticware (tips and tubes) and generates less chemical waste than any other commercial extraction system .prepGEM® features:• Thermostable enzyme & optimised buffer• Simple two step temperature profile (75°C

& 95°C)• DNA ready for PCR, qPCR, SNP testing in

20 min• No overnight incubations or centrifugation

required• Compatible with PCR or detection reagents

Material and methods

To demonstrate the performance of the prepGEM® kit for the type of hetero- and homozygosity assays commonly required in transgenic mouse facilities, 9 male and 7 female 3 month old mice were gender-tested after scheduled culling . DNA was extracted using the standard procedure for prepGEM®Tissue (1 µl prepGEM, 10 µl Buffer Gold, 89 µl H2O, incubate 75°C for 15 minutes and 95°C for 5 minutes). From the supernatant, 5 µl was added to a PCR mix and amplified using the primers described by Clapcote and Roder (2005):FORWARD 5’-CTGAAGCTTTTGGCTTTGAG-3’, REVERSE 5’-CCGCTGCCAAATTCTTTGG-3’These primers generate amplicons from the Jarid1c (Y-chromosome) and Jarid1d (X-chromosome) loci of 331 and 302 bp in males and a single band of 331 bp in females .

To evaluate the performance of the prepGEM® Tissue kit against similar commercially available kits, the same diagnostic assay was used on DNA templates generated using two other quick DNA-extraction kits cited here as Q and E . The procedures for these kits were followed according to manufacturers’ specifications, and where necessary, amplification was performed using the manufacturer-provided amplification kit .

qPCR reagentsIn order to demonstrate the ability of the extracted DNA to perform with PCR reagents from different vendors, three different qPCR methods were used: • PerfeCTa® SYBR® Green FastMix®

(Quanta Bioscience)• Platinum® SYBR® Green qPCR SuperMix-

UDG with Rox (Invitrogen) • Home-brew reagent mix with AmpliTaq®

DNA polymerase with Buffer II (Applied Biosystems) as follows: 1X Buffer II, 4 mM

MgCl2, 0,2 mM primers, 0,1 mM dNTPs, 0,24 mg/ml BSA, 1:200 SYBR, 0,04 mM ROX, 0,02 U Amplitaq

Cycling conditions were as follows: 95 °C, 2 mins; {95 °C, 30 s; 60 °C 30 s; 72 °C 30 s } x 40; Dissociation curve .

Results and discussionAmplification products were visualised using agarose gel electrophoresis and the yield and levels of inhibition determined by real-time qPCR . Validation of prepGEM® Tissue in conjunction with gender determining PCR can be seen in Figure 1 . A doublet was seen for all male animals and single bands for the females . The one exception had been incorrectly sexed by the anogenital distance (AGD) method .

Extraction comparisons

To determine comparative performance of the prepGEM® Tissue kit against commercially available kits with procedures similar to prepGEM® Tissue, six mouse tail tip samples were extracted using prepGEM®and two other rapid DNA extraction kits .

Again, the Jarid1 locus was amplified with either Platinum Taq (Invitrogen, Carlsbad) or the optimised PCR reagents supplied with the extraction kit .

Figure 1. Three month old culled mice tail tips extracted with prepGEM® Tissue and analysed on a 1,5% agarose gel (10 µl of PCR product was run without clean-up). Amplification of the Jarid1 locus from sexed male and reportedly female mice. * Incorrect determination by AGD in lane 17. Lane 1: 1kb+ ladder. Lane 19: Negative control.


This is demonstrated by the lower end-point fluorescence and slope of the plots when the Quanta and Homebrew reagents are used, and the failure with the Invitrogen qPCR reagents

ConclusionsThe prepGEM® Tissue kit offers researchers the tools for a rapid, robust and reliable DNA extraction that generates DNA templates that outperform other rapid commercial kits in both standard and quantitative PCR assays . Kit Q routinely gave lower DNA yields and although Kit E performed adequately using the vendor’s own reagents, it underperformed when alternative reagents were used . This

result suggests that the kit would have less utility than prepGEM® Tissue which offers researchers the flexibility to choose their own diagnostic reagents according to need . The likely reason for the improved performance of the prepGEM® Tissue kits is that the high temperature proteinase releases DNA from the sample without requiring chemical denaturants such as alkali, chaotropic salts or detergents . These substances can carry over into downstream reactions and cause reduced performance .

Reference

Clapcote S. J. and Roder J. C. (2005) Simplex CR assay for sex determination in mice. Biotechniques 38: 702-70.

Figure 3. qPCR from the three different extraction methods: prepGEM® (Blue), Kit Q (Red) and Kit E (Black). The assay was performed using three different qPCR mastermixes: Quanta PerfeCTa® SYBR® Green FastMix®(Top), Invitrogen Platinum® SYBR® Green pPCR SuperMix-UDG with Rox (Middle) and a Home-brew reagent mix with AmpliTaq® DNA polymerase with Buffer II (Applied Biosystems).


PCR products were visualised by agarose gel electrophoresis (Figure 2) . The prepGEM®Tissue kit yielded readily-amplified DNA with no migration distortion or smearing; kit Q showed a reduced yield of PCR product, as determined by band intensity . Kit E showed better yield of PCR using the manufacturer’s optimised PCR reagents but the high-molecular weight smearing above the PCR amplicon could compromise quantification .

Quantification of DNA Yield using different qPCR reagents

In order to determine the absolute yield of each extraction kit, qPCR was performed using three different qPCR methods (see materials and methods) . Standards were generated using a Qiagen DNeasy Kit and quantified with a 260/280 nM absorbance assay . The results of the qPCR reactions, performed on an Eppendorf epgradient S can be seen in Figure 3 . Yield from each DNA prep method was calculated . The average Yields were:• prepGEM® Tissue: 98,0 ng / mg of tissue from

2 mm tail tips .• Kit E samples: 0,34 ng / mg of tissue from 5 mm

tail tips .• Kit Q samples: 2,87 ng / mg of tissue from 5 mm

tail tips .In addition to the differences in the yields, it is notable from the plots that Kit E produced DNA which was inhibitory to the PCR .

Figure 2. Three month old culled mice tail tips extracted and the Jarid1 locus amplified. prepGEM® Tissue lanes 2-7, amplified with Platinum Taq; lanes 8-14 kit Q, amplified with manufacturer’s optimised PCR reagents; lanes 15-20 with kit E, amplified with Platinum Taq, and analysed on a 1,5% agarose gel (10 µl of PCR product was run without column clean up. Lane 1: 1kb+ ladder (Invitrogen)


BDH Prolabo Electran® covers the whole range of each of which is optimally specified for particular

Take advantage of our

HAPPY BIRTHDAY offer!

BDH Electran® DNA grade agaroseSee the VWRbioMarke shop for details

2009 is a very exciting year for our BDH Prolabo brand – the BDH part is 100 years old and Prolabo is now over 106 years old! We were very proud to merge these two fine brands in 2007 combining the best quality and price competitive characteristics of existing BDH and Prolabo products yet still retain our life science grade basics that have proven so useful to life scientists with its guarantee of reliability and high performance.


f agarose, particular applications

The BDH Electran® range of agaroses has been steadily improved and expanded in response to the growing demands of the newer techniques emerging in molecular biology.

We can now offer a wide variety of BDH Electran®

agaroses, each of which is optimally specified for particular applications.

ALL BDH agaroses are DNase/RNase FREE!

Agarose DNA Grade (100 bp – 23 kb)Agarose DNA Grade Electran® agarose is the ideal multipurpose agarose for any DNA or RNA application . Wide resolution range: 100 bp to 23 kb . Ideal for separation of DNA fragments larger than 1000 bp .

Agarose DNA Pure Grade (100 bp – 23 kb)Agarose DNA Pure Grade ensures reliable digestion and ligations from recovered DNA or RNA fragments from 100 bp to 25 kb . It is particularly suitable for both preparative and analytical separation procedures with DNA fragments ≥1000 bp. Gives low background after ethidium bromide staining .

Agarose High Resolution Plus (20 bp – 800 bp)Agarose High Resolution Plus offers twice the resolution capability of standard agarose . It allows separations traditionally performed on polyacrylamide to be carried out on agarose . It is possible to separate DNA fragments and PCR products with as little as 2% difference in base pairs in the range between 20 bp and 800 bp . Agarose High Resolution Plus gels of 2% and 4% gives approximate the resolution of polyacrylamide gels of 4% to 8% .

Agarose High Resolution (50 bp – 1 kb)Agarose High Resolution is the ideal choice for separating and resolving PCR and RT-PCR fragments . Superior resolution of small fragments between 50 bp and 1 kb .

Agarose High Resolution Low Melting (50 bp – 1 kb)

Agarose High Resolution Low Melting is a low melting temperature agarose which provides optimal separation and resolution of PCR and RT-PCR fragments . Fine resolution between 50 bp and 1 kb .

Agarose Wide Range Low Melting (200 bp – 25 kb)Agarose Wide Range Low Melting is a low melting temperature agarose (≤65 ºC). Confidently resolve fragments from 200 bp to 25 kb prior to PCR, cloning, digesting, or sequencing in the presence of re-100 g melted agarose, without additional purification steps . Ideal resolution of DNA fragments > 1000 bp

Agarose Megabase DNAAgarose Megabase DNA is ideal for separating very large DNA fragments or doing pulsed field gel electrophoresis (PFGE) and associated techniques for separation of chromosomal DNA . Capable of rapid separation of large DNA from 30 kb to 50 kb by horizontal electrophoresis or 50 kb to 10 Mb for PFGE .

Description Qty/Pk Cat. No.

Agarose DNA Grade Electran®

100 g 43879 2U

250 g 43879 3H

500 g 43879 4L

1 kg 43879 5A

Agarose DNA Pure Grade Electran®

25 g 44366 4V

100 g 44366 3U

250 g 44366 5W

500 g 44366 6A

Agarose High Resolution Plus Analytical Electran®25 g 43747 2D

100 g 43747 3E

Agarose, High Resolution Electran®25 g 43655 2V

100 g 43655 3W

Agarose High Resolution Low Melting Electran®

25 g 43712 2H

100 g 43712 3Y

1 kg 43879 5A

Agarose Wide Range Low Melting Electran®25 g 44415 2G

100 g 44415 3H

Agarose Megabase DNA Electran® 100 g 44416 3J



GelRedTM & GelGreenTMEnvironmentally safe and ultra-sensitive nucleic acid gel stains for replacing EtBr

GelRed and GelGreen are a new generation of fluorescent nucleic acid gel stains designed to replace the highly toxic ethidium bromide (EtBr). Developed by scientists at Biotium, GelRed and GelGreen are superior to EtBr and other EtBr alternatives by having a combination of low toxicity, high sensitivity and exceptional stability.

EtBr has been the predominant dye used for nucleic acid gel staining for decades because of its low price and generally sufficient sensitivity . However, EtBr is a highly mutagenic material . The safety hazard and costs associated with decontamination and waste disposal can ultimately make the dye expensive to use . For this reason, alternative gel stains, such as SYBR® dyes, have become commercially available in recent years . Although these alternative dyes have reduced mutagenicity, they often have to sacrifice other aspects of the dyes . For example, SYBR® Safe has very limited sensitivity while SYBR® Green and SYBR® Gold are much less stable than EtBr . SYBR®dyes also enter cells rapidly to stain mitochondria and nuclear DNA, making it more likely for the dyes to be toxic at high enough concentrations . Indeed, SYBR® Green I is known to strongly potentiate mutation caused by UV light or another mutagen (Ohta, et al . Mutat . Res . 492, 91(2001)) .

To make GelRed and GelGreen safe, scientists at Biotium used a novel yet very simple concept: reducing genotoxicity by preventing the dyes from entering living cells . We believe that a DNA-binding dye can be made non-mutagenic or substantially so by denying its chance to be in contact with

genomic DNA in living cells . Thus, we engineered the chemical structures of GelRed and GelGreen such that the dyes are incapable of crossing cell membranes . The Ames test confirmed that GelRed and GelGreen are non mutagenic at concentrations well above their working concentrations used for gel staining . Furthermore, environmental safety tests showed that GelRed and GelGreen are nonhazardous and nontoxic to aquatic life . As a result, GelRed and GelGreen can be disposed of down the drain or in regular rubbish .

GelRed and GelGreen are highly sensitive either as precast gel stains or post gel stains . Designed primarily for use with a 312/302 nm UV transilluminator, GelRed is much more sensitive than EtBr, and at least as sensitive as or brighter than SYBR® Gold in post gel staining . Unlike SYBR®Gold, GelRed can also be used as a highly sensitive precast gel stain . GelGreen is developed to meet the needs of researchers who use a 488 nm laser-based gel scanner or a Dark Reader that uses a visible blue light for excitation . GelGreen is spectrally similar to SYBR® Safe, but is far more sensitive than the latter .

Another major advantage of GelRed and GelGreen is their remarkable stability . You can handle the two dyes

Figure 1. Comparison of GelRedTM and ethidium bromide (EtBr) in precast gel staining using 1% agarose gel in TBE buffer. Two-fold serial dilutions of 1 kb Plus DNA Ladder from Invitrogen were loaded onto each gel in 4 lanes in the amounts of 200 ng, 100 ng, 50 ng and 25 ng, respectively, from left to right. Gels were imaged using a 300 nm transilluminator and photographed with an EtBr filter and Polaroid 667 black and white print film.

Figure 2. Comparison of GelRedTM and ethidium bromide (EtBr) in post gel staining using 1% agarose gel in TBE buffer. Two-fold serial dilutions of 1 kb Plus DNA Ladder from Invitrogen were loaded onto each gel in 4 lanes in the amounts of 200 ng, 100 ng, 50 ng and 25 ng, respectively, from left to right. Gels were imaged using a 300 nm transilluminator and photographed with an EtBr filter and Polaroid 667 black and white print film.



Genomics

the same way you do with EtBr . This means that the dyes are perfectly stable in water at room temperature for long-term storage, and they can be microwaved for making precast gels . Both dyes are also very photostable, permitting their use under normal room light without exercising special precaution .

Request your

personal sample at

[email protected]

200 300 400 500 600 700Wavelength (nm)

GelRed ExcitationGelRed Emission

GelGreen Excitation Gel Green Emission

Figure 3. Comparison of GelGreenTM and SYBR® Safe in precast gel staining using 1% agarose gel in TBE buffer. Two-fold serial dilutions of 1 kb Plus DNA Ladder from Invitrogen were loaded onto each gel in 4 lanes in the amounts of 200 ng, 100 ng, 50 ng and 25 ng, respectively, from left to right. Gels were imaged using a 302 nm transilluminator and photographed with a SYBR®filter using the GelDoc-ItTMimaging system from UVP.

Figure 4. Comparison of GelGreenTM and SYBR® Safe in post gel staining using 1% agarose gel in TBE buffer. Two-fold serial dilutions of 1 kb Plus DNA Ladder from Invitrogen were loaded onto each gel in 4 lanes in the amounts of 200 ng, 100 ng, 50 ng and 25 ng, respectively, from left to right. Gels were imaged using a 254 nm transilluminator and photographed with a SYBR filter and Polaroid 667 black and white print film.

Figure 5. Normalised excitation and emission spectra of GelGreenTM (green) and GelRedTM (red) in the presence of dsDNA in PBS buffer.

0

200

400

600

800

1000

1200

0 0.1 0.25 0.5 1 2.5 5 7.5 10 25 50

Dose (ug/plate)

Nu

mb

er o

f C

olo

nie

s

EtBr GelGreen GelRed

Concentration range used in gel staining

* **

Figure 7. Comparison of mutagenicity among GelRedTM, GelGreenTM and EtBr. Ames tests performed in Salmonella strain TA98 with S9 metabolic activation. * Indicates EB not tested at these concentrations.

Figure 6. HeLa cells were incubated with 1X of SYBR® Safe, GelGreenTM or GelRedTM. Images were taken following incubation for 30 min. SYBR® Safe entered into cells rapidly as evident from the bright green nuclear staining. However, GelRedTM and GelGreenTM were unable to cross cell membranes as shown by the lack of any fluorescence staining.


An easy determination of the antioxidant power of with Thermo Scientific Multiskan FC Microplate

Oxidative stress has been demonstrated to be involved in many diseases including atheroschlerosis, Parkinson’s disease and cancer . Oxidative stress is caused by an imbalance between the production of reactive oxygen and a biological system’s ability to detoxify the reactive intermediates, or easily repair the resulting damages .

The most common forms of reactive oxygen are free radicals like superoxide anions, hydroxy radicals and alkoxy or peroxy radicals, which start chain reactions that damage cells . Antioxidants are molecules capable of slowing down or preventing the oxidation of other molecules, therefore protecting cells from the oxidation damages . Antioxidants can remove free radical intermediates, and inhibit other oxidation reactions by being oxidised themselves .

Commonly known examples of antioxidants are vitamins E and C, uric acid, carotenes and glutathione . This paper describes how the total antioxidant capacity of the unknown samples can be measured with the photometric assay . The assay is based on the reduction of bivalent copper ion, Cu2+, to Cu+ by the antioxidants present on the sample . This reduced form of copper is then selectively bound to chromogenic compound to form a complex that has strong absorbance at 490 nm .

The assay is calibrated by using known concentrations of standard antioxidant .

Materials and methods

The beverage samples used to measure antioxidant power were 100% natural orange and apple juices as well as green, black and white tea . Antioxidant power was determined with a photometric Total Antioxidant Power kit and clear flat bottom 96-well Thermo Scientific Immulon 1B microplates (Cat . No . 735-0461) . The assay was performed according to the kit instructions, except that vitamin B analogue TroloxTM ((±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) (Calbiochem, Cat . No . 648471-500) was used as a second standard for calibrating the assay in addition to the uric acid standard of the kit . Beverage samples were tested with three dilutions: undiluted, 1:5 and 1:10 dilutions. A 200 μl aliquot of each standard and sample dilution was added as duplicate into a 96 well microplate . Then, the background absorbance of the plate was measured at 492 nm with Multiskan FC controlled by Thermo Scientific SkanIt® Software. After that, 50 μl of the Copper Solution was added . The assay plate was then incubated for three minutes at room temperature and the reaction was stopped by adding 50 μl of the Stop Solution. The absorbance was then measured again at 492 nm immediately .

Background absorbance of each well before the addition of the Copper Solution was subtracted from the final absorbance values and linear standard curves of uric acid and Trolox were created based on these subtracted absorbances . The antioxidant power of each unknown sample was then resolved from the standard curves as uric acid and Trolox equivalents (uric acid or Trolox concentration that has equivalent antioxidant efficiency as the unknown sample) .

Results and discussionThe subtraction of background absorbance, creation of the standard curves and calculation of the concentrations of the unknown samples were performed with the automatic calculations of the SkanIt Software . The resulting standard curves of uric acid and Trolox are shown in figure 1 .

Assay robustness was analysed using Z-prime calculation . The resulting values are shown in table 1 .

This paper shows how antioxidant power of different beverage samples can be easily and rapidly tested with a photometric assay using Thermo Scientific Multiskan® FC microplate photometer. With Multiskan® FC reader it is possible to measure antioxidants with high sensitivity and assay robustness. The assay is also very fast to perform, the total time for the complete assay is around 5 - 10 min.


SampleDilution factor

Uric acid equivalence

(mM)

Trolox equivalence

(mM)

Apple juice1 3,1 2,65 6,0 5,2

10 6,5 5,6

Orange juice1 3,1 2,75 7,9 6,8

10 8,4 7,2

Black tea1 2,9 2,55 8,0 6,9

10 8,6 7,4

White tea1 3,2 2,85 14,0 12,1

10 15,9 13,7

Green tea1 3,1 2,75 14,6 12,610 17,9 15,4

Extrapolation of the standard curve data was allowed to calculate the result values higher than the highest standard . These results are shown in table 2 . One striking observation from the results was the excellent precision of the reader with low level absorbances .

The background values measured before the dye addition had clearly below 0,001 absorbance unit standard deviation in the blank values (n = 46) . This exceptional low level precision improves the assay sensitivity because absorbance values very close to background become statistically relevant and can be used for the real measurements .

Both standard curve analysis and Z-prime analysis show that this antioxidant power assay can be performed perfectly with the Multiskan FC reader . The standard curves show complete linearity over the whole concentration range and values prove that

the assay can be done even below the concentration range recommended in the kit instructions .

When results of unknown beverage samples were analysed, first thing noticed was that undiluted samples clearly contain some component disturbing the assay . This is seen from the calculated Trolox and uric acid equivalents that were always remarkably lower with undiluted samples than with diluted samples . Instead, samples with 1:5 and 1:10 dilution factors behaved as expected so they gave identical results when dilutions were taken into account .

This shows that it is recommended to always test such heterogenous samples with a dilution series instead of using only one sample concentration . Green tea and white tea were shown to have clearly the highest antioxidant power in this assay . This was to be expected because green and white tea have been well known to include quite strong antioxidants . Orange juice and black tea had quite similar antioxidant levels and apple juice was the one having the least antioxidant power of these five beverages . These values are quite typical for the beverages analysed here . Large variations in the antioxidative power can anyhow be observed when other types of green, black or white teas, or juices, are used .

For further information about Multiskan FC, please contact your local VWR sales office .

of beverage samples Photometer

Figure 1. Uric acid and Trolox standard curves of the assay. The curves show perfect correlation between the amount of known antioxidant and resulting absorbance. Trolox is known to be a stronger antioxidant than uric acid. The absorbance difference between each uric acid and Trolox standard remains constant (~15%) over the whole concentration range.

Concentration (mM) Z-prime Uric acid Z-prime Trolox

0,03125 0,40 0,880,0625 0,93 0,920,125 0,81 0,850,25 0,95 0,920,5 0,90 0,991,0 0,99 0,992,0 0,97 0,93

Table 1. Values of the assay. The values are close to 0.9 – 1.0 (except the lowest uric acid concentration) which proves the excellent robustness of the assay.

Table 2. Antioxidant power of analysed beverage samples given as Trolox and uric acid equivalents.

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


Large scale suspension cell growth and secreted the Corning® HYPERFlask® cell culture vessel

The Corning HYPERFlaskcell culture vessel was developed to meet increased needs for larger amounts of cells and secreted products such as antibodies by the pharmaceutical industry.

With the same overall dimensions as the standard T175 flask, the HYPERFlaskvessel is comprised of ten individual gas permeable layers, which allow for approximately ten times the total growth surface of a T175 . As previously shown (see A Novel Flask Design for High Density Cell Culture), this increased growth area translates to ten times the total cell yield for adherent cell lines . We show here that similar yields are also achieved with both serum-free and serum-dependent suspension cell lines . Growth comparisons of CHOS and MH677 cells in the HYPERFlask vessel relative to a standard T175 flask showed no difference in cell density or viability . Further, the production of monoclonal antibodies (mouse IgG2a) by hybridoma cells was equivalent, per cm2 of growth surface, in both flasks, resulting in a 10-fold greater yield of cells from the HYPERFlask vessel .

Materials and Methods CHOS cells cultured in serum-free CDCHO medium supplemented with 20 mM LGlutamine and 1X hypoxanthine thymidine solution were seeded into T175 (Cat . No . 734-1733) and HYPERFlask vessels (Cat . No . 734-4009) at a density of 2,5 x 105

cells/ml and volume of 0,326 ml/cm2 . The vessels were placed in a humidified incubator at 37 °C with 5% CO2 for 96 hours . Cell suspensions were collected and counted with a Z2 series particle counter . Each experiment was performed in triplicate and replicated a total of three times . For antibody production studies, hybridoma cells were seeded into T175 flasks and HYPERFlaskvessels at a density of 5 x 104 cells/ml and volume

Figure 2. Average MH-677 cell density at 24-hour time points for 96 hours in T175 and HYPERFlask™ cell culture vessels. Data respresent an average of three independent studies each done in triplicate (±S.E. indicated by error bars; p>.05).

Figure 1. Total CHOS cell construction after 96 hours in T175 and HYPERFlask™cell culture vessels. Data represent an average of three independent studies each done in triplicate (±S.E. indicated by error bars; p>.05).

of 0,326 ml/cm2 in IMDM supplemented with 10% foetal bovine solution . The vessels were incubated at 37 °C and 5% CO2 for 96 hours . One millilitre samples from each flask were collected and counted every 24 hours . Cell viability was also determined with a Nova BioProfile Flex via trypan blue exclusion . At the end of the 96 hour growth period, an additional sample was taken from each flask and centrifuged at 270 RCF for 7 minutes . The supernatant was collected and used to determine antibody production following an ELISA kit protocol . The experiment was replicated a total of three times, each performed in triplicate .

Results The CHOS cell line is a commercially available serum-free adapted clone of CHO cells and was selected to demonstrate the performance of suspension cells in serum-free media in the HYPERFlask vessel . Results showed equivalent cell densities, resulting in a 10-fold greater yield of cells from the HYPERFlask vessel . (Figure 1, see reverse side) . As a further example of suspension cell growth in the HYPERFlask vessel, hybridoma cells were seeded in T175 and HYPERFlask vessels, and cell density and viability were determined at

24-hour time points for 96 hours . Data showed equal growth rates for each vessel (Figure 2), with average viability greater than 95% for both (data not shown) .

As a hybridoma cell line engineered for the production of monoclonal antibodies, these cells serve as a model for relative secretory production . ELISA quantification of antibody production shows equal concentrations of mouse IgG2a per cm2generated in the HYPERFlask vessel compared to the T175 control (Figure 3) . With ten times the total growth area of a T175, net IgG2a production in the HYPERFlask vessel was also tenfold (Figure 4) .

Figure 4. Total mouse IgG2a production after 96 hours in T175 and HYPERFlask™ cell culture vessels. Data respresent an average of three independent studies each done in triplicate (±S.E. indicated by error bars; p>.05).


secreted product yields using

Figure 3. Average mouse IgG2a production/cm2 after 96 hours. Data respresent an average of three independent studies each done in triplicate (±S.E. indicated by error bars; p>.05).



SuperSpinner D1000 – disposable bioreactor for efficient cell cultivation

Growing cells in an artificial environment like a bioreactor vessel requires the delicate balance of several parameters. Not only are the cells meant to grow to relatively high numbers; usually they are also supposed to produce a particular target protein (e.g. an antibody) in high amounts – these are all processes requiring a high metabolic activity, which is directly linked to a high demand for oxygen! This is why the classic bioreactor design usually includes sophisticated technology and instruments to monitor and control the oxygen saturation of the cell culture media. Lab scale devices in contrast generally lack any such equipment. Oxygen uptake into the media is restricted to surface aeration and thus oxygen supply to the cells often is a limiting factor. As a result the optimum in cell numbers and production kinetics can not be achieved.

The SuperSpinner D 1000 is a disposable stand-alone cultivation unit, which was developed for efficient cell cultivation . It is optimised for best possible oxygen supply without using sophisticated equipment and high stirring rates . It is easy to use and enables cost-

efficient, laboratory-scale cell cultivation with a recommended maximum working volume of 1l . litre . The key feature of the SuperSpinner D 1000 is an integrated membrane aeration and agitation system, which allows for optimal gas transfer into the media (Figure 1) . A hollow-fibre membrane is wound around a stirrer bar with an iron core at the tip; agitation is achieved by a magnetic drive unit . A membrane pump complements the setup; it feeds ambient air through a sterile filter into the hollow-fibre membrane . Oxygen and carbon dioxide pass into the cell suspension by passive diffusion . The multiple windings of the hollow fibre represent an enormous increase in the (active) aeration surface . Thus, oxygen is not limiting the cell growth, which results in higher productivity compared to traditional spinner flasks . The complete set-up can be placed inside regulated incubator if special gases are required .

The SuperSpinner D 1000 is delivered preassembled and single packed in a sterile pouch . A special feature is the sampling system equiped with a clave adapter which facilitates the drawing of samples at any time in place . The cultivation flask itself (1 l total volume) is compatible with standard rotors,

Figure 1. The SuperSpinner D 1000 disposable bioreactor



hence the cell suspension can be centrifuged within the cultivation vessel . If the content of the SuperSpinner D 1000 is intended to be used as seed for a larger bioreactor, the cultivation broth can be transferred directly into the bioreactor vessel by using the thermoplastic C-Flex R symbol tube and a BioWelder® / BioSealer® System .

This short note describes the cultivation of Spodoptera frugiperda Sf9 cells and Chinese hamster ovary (CHO) cells, respectively, in the SuperSpinner D 1000 .

CHO cells in proCHO5 medium (0,8 l working volume) were cultivated at 37 °C in a CO2 incubator with a CO2 saturation of 5% . The starting cell density was 1 × 106 viable cells/ml . The stirrer speed was set to 60 rpm . A membrane air pump served as aeration system . Samples were drawn once a day via the clave adaptor of the sample port to check for cell count and viability; the bioreactor was not

removed from the incubator during this procedure . The cultivation of CHO cells in the SuperSpinner D 1000 resulted in a maximum viable cell number of 4 × 106 cells/ml after 3 days of cultivation . The maximum cell density attained in a traditional spinner flask run in parallel was less than 2 × 106viable cells/ml . (Figure 3)

In another experiment Sf9 cells (non-transfected) in Sf-900 III medium (serum free, 1 l working volume) were cultivated at 27 °C in an incubator, with the stirring rate of the magnetic drive unit adjusted to 75 rpm . The cells were aerated with 0,1 v .v .m . (gas volume/liquid volume/minute; gassing rate was adjusted by using the rotameter system of the incubator) . The starting cell density was 2 × 106 viable cells/ml . Samples from the inside of the vessels were acquired like described above and cell count and viability were determined . A maximum viable cell number of 1,66 × 107 cells/ml was achieved at day 4 after inoculation (Figure 3) . Cultivation in a traditional spinner flask under the same conditions resulted in a cell density of 4–5 × 106 viable cells/ml (personal communication; R . Eibl) .

It can be summarised that the cultivation of Sf9 cells in the SuperSpinner D 1000 resulted in a maximum viable cell number more than 3 times higher compared to cells grown in a traditional spinner flask, operating with surface aeration (personal communication; R . Eibl) . When cultivating CHO cells in the SuperSpinner D 1000, a two-fold increase over the numbers in a classic spinner flask could be observed .

In another line of experiments (results not shown) the SuperSpinner D 1000 was used to cultivate murine hybridoma cells . In these studies, a maximum cell density of 3,16 × 106 viable cells/ml was achieved, which is ~2,25 times higher than in traditional spinner flasks (personal communication; C . Schwiebert, InVivo BioTech Services GmbH) .

ACKNOWLEDGMENTSThe author thanks I. Bauer, L. Lisica and R. Eibl of the Zurich University of Applied Sciences, and C. Schwiebert of InVivo BioTech Services GmbH, for support and providing the data.

Figure 2. Batch cultivation of Sf9 cells in Sf-900 III medium at 27 °C, 75 rpm and 0,1 v.v.m. Samples were collected daily and analysed for viable cell number and viability.

Figure 3. Batch cultivation of CHO cells in proCHO5 medium at 37 °C, 60 rpm, with aeration via a membrane pump and at 5% CO2 saturation in SuperSpinner D 1000 and a traditional spinner flask. Samples were collected daily and analysed for viable cell number and viability.


BRANDplates® microplates Comparison of adsorption capacity between BRAND immunoGrade™ microplates and different competitors

Modern research and development in the field of immunological applications like ELISA, RIA, FIA, etc. is not possible without high-quality plastic disposables. To ensure reproducible results a consistent quality is needed. In addition to the selection of high quality raw materials with excellent optical characteristics a very homogeneous physical/ chemical process leads to a variety of BRANDplates® with different binding properties. All BRANDplates®

microplates can be stored at room temperature and are supplied free from endotoxins, DNA, DNase, RNase and cytotoxic substances.

BRAND produces three different surfaces for immunological applications:

1 . immunoGrade™: basic immunological surface, optimised for the immobilisation of IgG and other molecules with hydrophilic and hydrophobic regions .

2 . hydroGrade™: Strongly hydrophilic surface, optimised for hydrophilic molecules like glycoproteins, nucleic acids and proteins with hydrophilic character .

3 . lipoGrade™: Strongly hydrophobic (lipophilic) surface for immobilisation of biomolecules with predominantly hydrophobic areas like lipoproteins or peptides .

This is the comparison between BRANDplates®immunoGrade™ and some of the high binding surfaces of direct competitors using a direct antibody binding assay .

Materials and methods

1. Chemicals and reagents• TMB (3,3´, 5,5´-Tetramethylbenzidine, Merck

KGaA)• Hydrogen peroxide 30% (Merck KGaA)• Tween 20 (Merck KGaA)• Polyclonal Rabbit AntiHuman IgA-HRP conjugate,

ref . P0216 (DAKO North America, Inc .)• All other reagents were of the highest purity

commercially available .

2. 96 well Microplates• BRANDplates® pureGrade™ (non-treated) and

immunoGrade™ (BRAND GMBH + CO KG, Germany)

• Competitor A • Competitor B • Competitor C

3. Direct antibody adsorption assayThe idea behind the assay is the detection of the binding capacity of the different surfaces using an antibody linked with a peroxidase .

HRP: horse radish peroxidase (enzyme that uses H2O2 as substrate) TMB: (3, 3’, 5, 5’-tetramethylbenzidine) is the most commonly used and most sensitive substrate for molecules labelled with the enzyme horse radish peroxidase (HRP) . In presence of HRP and H2O2 TMB is oxidised to a deep blue product . By addition of acids the product is modified to a yellow molecule with a 2 – 4 times greater molar extinction coefficient than the blue product . Detection is at 450 nm .



Results : Direct antibody adsorption assay

This assay allows the determination of the quantity of protein (IgA) bound on different modified surfaces using the oxidation of TMB. The comparison of BRANDplates® microplates immunoGrade™ with untreated PS surfaces and competitors high binding plates shows that the new BRANDplates® surface leads to slightly higher adsorption.

Conclusion

The antibody adsorption on BRANDplates®immunoGrade™ surface was compared with high quality high binding surfaces from three competitors.

The new immunoGrade™ surface shows a higher immunoglobulin adsorption compared with competitive products.

1. Add to a 96-well microplate 100 µl of a rabbit IgA HRP-conjugate with dif-ferent concentrations (1:4000 to 1: 1 024 000) in 100 mM carbonate buffer pH 9,6

2. Seal plate with an adhesive film and incubate at room temperature for 12 h

3. Wash 3 times with0,15 M PBS pH 7,2containing 0.05% Tween20

4. Add 100 µl substrate solution (5 % TMB/ 0,04 % H2O2)

5. Stop reaction with 150 µl H2SO4 and read O.D. at 450 nm using plate reader (SPECTRAmax 384 plus, Molecular Devices, Corp., USA)

Results of the comparison using a direct IgA adsorption assay


Ten thousand cells were seeded onto TC-treated, Competitor C, or 384 well black/clear BD PureCoat amine plates and grown under serum-free conditions for 20-24 hours. The cells were then washed (on an Embla cell washer) two times with Hank’s buffered salt solution containing 10 mM Hepes, loaded with calcein AM for 1 hour and read on an EnVision®(PerkinElmer) plate reader. Before and after wash images indicate that cells remain attached on BD PureCoat amine surface and are washed off the other surfaces that were tested.

BD PureCoat Cultureware Where life thrives on the surface

TC-Treated Com

petitor C BD PureCoat Am

ine

Before Wash After Wash

BD PureCoatTM surfaces are a novel family of chemically-defined, animal-free cell culture surfaces designed to enhance cell performance. A proprietary thin-film coating technology produces a uniform, functionalised surface that provides a highly controlled environment for cell culture applications. Both BD PureCoat amine, a positively charged surface, and BD PureCoat carboxyl, a negatively charged surface, are proven to provide improved attachment, increased proliferation, enhanced recovery from freeze-thaw, and improved differentiation for a broad range of cells that exhibit poor attachment properties in serum-free or serum-reduced conditions (e.g., primary cells, transfected cells, and fastidious cell lines). In drug discovery assays, when using transfected cells and commercially prepared division arrested cell lines, both surfaces maintain cell monolayers during vigorous liquid handling manipulation. See Table 1 for a list of cell types that have been successfully cultured on BD PureCoat Amine and Carboxyl.

Improved post-wash adherence of Eco-Pack2-293 cells (a transformed HEK-293 cell line) to BD PureCoat amine

Advantages of BD PureCoat Amine and Carboxyl Surfaces

• Optimal performance. Surface technology enhances attachment, proliferation, and recovery post-thaw for a variety of cells with poor attachment properties – mainly primary cells, transfected cells, and fastidious cell lines in serum-free or serum-reduced conditions .

• Lot-to-lot consistency. Both surfaces are highly consistent from lot-to-lot. Each lot is quality control tested and cell culture certified using an appropriate cell line .

• Delivering choice. Surface chemistries are available on a variety of cultureware for preparative cell culture and drug discovery assays .



A. Early passage LnCAP cells cryopreserved in growth medium (RPMI-1640, 10% foetal bovine serum) and 5% DMSO were thawed in a 37°C water bath and immediately seeded onto 96-well black/clear TC-treated, Competitor C, or BD PureCoatTM carboxyl plates at 16,000 cells/well. After an overnight incubation, cultures were observed and images captured at 100X magnification. Results from a representative experiment show that a greater number of LnCAP cells are evenly attached on BD PureCoat carboxyl versus TC-treated or Competitor C plates.

B. This increase in cell attachment and proliferation was significantly greater (p


Biochrom is a company that produces and supplies cell culture media and quality animal sera . Established in 1981, the company manufactures and distributes the full range of products related to mammalian cell culturing technique: standard cell culture media, complete media tailored for particular cell types and cell lines, custom formulations and animal sera, such as Foetal Bovine Serum (FBS) . All sera are pre-screened prior to acceptance; only those lots that meet strictly quality control requirements are accepted for further processing and subsequent sale to customers . Serum products are tested for sterility, adventitious viral agents, physico-chemical parameters, and biological performance .

The manufacturing processes are designed for the specific needs of customers engaged in biotechnological research, development and biopharmaceutical production .

Biochrom specialises in meeting customers‘ specifications and regulatory concerns, to facilitate the manufacture of custom media, consistent with Good Manufacturing Practice (GMP) . The media are produced in a strictly controlled environment; chemicals used conform, where applicable, to grade standards of European Pharmacopoeia and US Pharmacopoeia .

Cell culture as a platform technology, and tissue engineering is becoming an important part in human health care . As part of this exciting development, Biochrom puts growing research efforts in the development of media suitable for applications such as somatic cell therapy, tissue replacement and organ transplantation, manufactured according to GMP standards .

Through in-licensing and numerous research projects with both universities and biotech companies, Biochrom is connected to intellectual resources specialising in today’s cell culture applications .

Manufacturing standardsBiochrom offers highest quality and competitive pricing at the same time; all manufacturing processes and strict quality systems enable and guarantee low endotoxin levels for liquid bulk products .

Biochrom follows Good Manufacturing Practice (GMP) guidelines: this requires validation, and documentation of all operational procedures, systems, functions, facilities, materials, and equipment . Products are prepared by an aseptic process for which each step has been validated to ensure that all liquids do meet the highest standards .

Biochrom has established Standard Operating Procedures (SOPs) for all its manufacturing and quality procedures . The company validates its equipment and processes on a regular level to ensure that they meet specific designed criteria in adaptation to internal and external quality assurance .

Audits are performed to verify that manufacturing and quality control functions are being properly executed .

Sterile Process Liquids, Buffers and Balanced Salts from Biochrom

Rapid advances in biopharmaceutical production have created a growing need for reliable sources of sterile process liquids delivered ‘just in time’ .

Biochrom’s production of bulk sterile liquids is based on its source of highly purifiedwater, that does meet the Water for Injection (‘WFI’) criteria . All commonly used buffers, balanced salts and cell culture media are available according to customer’s own specifications, manufactured under GMP, and ISO 9001 guidelines . Production incorporate a rigorous specification and approval process that includes screening of all incoming related raw material, this specifies Ph .Eur . grade components, and/or components of non-animal origin .

All Biochrom process liquids are intensively tested prior to QC release, and packed in containers ranging from 100, 500 and 1000 ml PET bottles, 100 ml to 50 l media bags or larger volumes of 200, 500 and 1000 l media bags, shipped out in appropriate stainless steel containers .

Welcome to a new VWRBioMarke partner



Pack (ml) Cat. No.

100 BCHRS0613500 BCHRS0615

Easy to use:

FBS-Superior from Biochrom

In order to even the usual variations of the biological characteristics of FBS batches, Biochrom offers with FBS Superior a standardised quality serum . Due to the addition of selected synthetic supplements, a continuous high level of quality is guaranteed .

Manage routine cell culture in the future without any need to test FBS batches, and use the Biochrom FBS that provides constant quality and excellent growth-enhancing characteristics .

On the next testing, also ask for the FBS Superior and convince yourself of the high quality and the benefits, which offers the FBS Superior .

• Once tested, the FBS provides a balanced constant growth profile

• Save further testing in the future• The costs for the FBS Superior are the same

compared to usual EU-approved FBS• FBS Superior is always available

Order a free sample today! Contact [email protected]

Here’s an example

of one of our key products!


Wheaton E-Z ExTraction Glass MicroTubes – glass is back!

Millions of tubes are sold every year into many compound storage / sample storage libraries around the world . Most of these small volume tubes are made of plastic while the remaining tubes are glass . Many people would agree that glass is better, however, the less expensive plastic version is often chosen . Some of the advantages of using glass tubes over plastic tubes are:

Glass microtubes Plastic microtubesType I borosilicate glass Various plastic resinsLow level of extractables Many leachablesNo gas permeability Gas permeableHigh chemical resistance Lower chemical resistanceLow sample adherence High sample adherence

Wheaton Industries, an industry leader in glass manufacturing for over 120 years, has come out with a new 96 well rack of Glass Microtubes that competes with plastic microtube pricing . These tubes are type 1 borosilicate glass and come with unique ceramic 2D bar codes or without bar codes . This opens new doors for companies that have a lot of time and resources invested into their samples, but could never justify the high costs for traditional glass microtubes .

Now companies can overcome problems with plastic microtubes when they need to store chlorinated compounds, acidic, basic and other corrosive chemicals with Wheaton E-Z Ex-Traction Glass MicroTubes . The long-term integrity of the samples stored in the Glass MicroTubes is improved

considerably . The precision Glass MicroTubes are priced very competitively without compromising quality and are offered at a small premium to the plastic tubes widely available in the market .

Applications and TraceabilityEZ-ExTraction Glass MicroTubes are ideal for use in Compound Storage Libraries, Critical Sample Handling Facilities, Cell Banks, Antibody Production Facilities, and in Pharmagenomics . Glass microtubes work well with expensive samples by havin

Documents

vwruk.vwr-cmd.com/ex/downloads/life_science/biomarke/0909/VWR_bioMa… · reproducible experimental data . ABgene® SuperPlate™ Genomics Figure 2. Competitor plate (left) may warp