2011 course on Molecular Diagnostic Automation - Part 1 - DNA Extraction
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Transcript of 2011 course on Molecular Diagnostic Automation - Part 1 - DNA Extraction
Nucleic Acid Extraction Automationin Molecular Diagnostic
part 1
Patrick MerelBiomedical Innovation Platform (PTIB), Pessac, France
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DNA and RNA from...
Blood, Plasma, Cells … Body Fluids
Nucleic Acid (NA) extraction in molecular diagnostic
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Quality of DNA/RNA is crucial for the PCR, RT/PCR steps
Extraction procedures often require centrifugation or filtration
Which are difficult steps to automate
Or, which require complex instrumentation
However, there are now numerous options for automation of NA extraction steps
Semi-automated protocols (microtiterplate based)
Automated protocols on robotic workstations
Dedicated and automatic workstations
But first, let’s go shopping for the perfect NA extraction technology...
NA Extraction Steps Automation
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Organic extraction is a conventional technique that uses organic solvents to extract contaminants from cell lysates.
The cells are lysed using a detergent, and then mixed with phenol, chloroform, and isoamyl alcohol.
The correct salt concentration and pH must be used during extraction to ensure that contaminants are separated into the organic phase and that DNA remains in the aqueous phase.
DNA is usually recovered from the aqueous phase by alcohol precipitation.
NA organic extraction
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NA Phenol-chloroform extraction
Equal volumes of a phenol:chloroform mixture and the aqueous DNA sample are mixed, forming a biphasic mixture.
The proteins will partition into the organic phase while the DNA (as well as other contaminants such as salts, sugars, etc.) remain in the aqueous phase.
It is then followed by precipitation with ethanol, as ethanol precipitates the nucleic acid.
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DNA extraction by Salting-Out
•Salting-out is another conventional technique where proteins and other contaminants are precipitated from the cell lysate using high concentrations of salt such as potassium acetate or ammonium acetate.
•The precipitates are removed by centrifugation, and the DNA is recovered by alcohol precipitation.
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Most nucleic acid purification techniques that use silica in combination with chaotropic reagents such as guanidine hydrochloride are covered by U.S. Patent No. 5,234,809, European Patent No. 0,389,063, and equivalents in other countries. It is commonly known as the "Boom" patents and is owned
by Biomerieux.
DNA Separation by Silica Adsorption
•This method is based on the selective adsorption of nucleic acids to a silica-gel membrane in the presence of high concentrations of chaotropic salts.
•Use of optimized buffers in the lysis procedure ensures that only DNA is adsorbed while cellular proteins, and metabolites remain in solution and are subsequently washed away.
•Ready-to-use DNA is then eluted from the silica-gel membrane using a low-salt buffer.
•No alcohol precipitation is required but filtration or combination of filtration+centrifugation procedures are needed.
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Mainly single tube based,but various options for
microtiterplate processing are now available
Centrifugation + filtration NA extraction
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Filtration + centrifugation NA extraction
Filtration only procedures available for single unit processing, and
microtiter plate based products for robotic handling.
Most of the time filtration procedures end with a centrifugation
step.
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Magnetic beads based NA extraction
High quality nucleic acids
Scalable
No centrifugation
No vacuum steps
Rapid purification
Reproducible12
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Dynal Dynabeads Technology
•Dynabeads a re super pa ramagne t i c , monosized polymer particles, providing a consistent, defined surface for the adsorbtion or coupling of various bioreactive molecules.
•The beads are magnetisable and super paramagnetic, meaning they are only magnetic in a magnetic field.•Due to this property, the beads can easily be resuspended when the magnetic field is removed. •The technology behind the beads was licensed to Dyno Industrier in 1980, which later formed Dynal Biotech together with Apothekernes Laboratorium in 1986.
Scanning Electron Microscopic pictures of the monosized Dynabeads® (A) and alternative
magnetisable particles from other suppliers (B-E). Courtesy of Mikal Heldal, University of Bergen,
Norway. 13
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๏Lysis and binding 5+5 min
๏Wash, Rinse 3x1 min
๏Elution 30 s RT
๏or 5 min 65C
๏Total @ RT <15 min
•Chaotropic soln (GTC) + silica magnetic particles
•Chaotropic solution, removes non-bound matter
•EtOH, removes GTC
•Water, removes EtOH
•Water or low salt buffer
Dynal Dynabeads Procedure
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Agencourt SPRI Technology
•The Agencourt SPRI (Solid Phase Reversible Immobilization) technology is a patented purification technology developed at MIT's Whitehead Institute (Hawkins, et. al., Nucleic Acids Res. 1995 (23): 4742-4743).
•Nucleic acids are immobilized onto paramagnetic microparticles (1µM magnetic carboxylate-modified base particles) using specific buffer conditions. Flexibility is added to the system through modification of the binding buffer, which alters the type and size of immobilized nucleic acid.
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Agencourt SPRI Technology
Lysis 30 min RTBind 5 min
Capture 15 minWashing1 2x10 minWashing2 2x6 min
Capture+Elution 2+10 min
Total @ RT 100 min16
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Magnetic Extraction Pros and Cons
Pros•Usually very fast procedures
•Robotic friendly•Rapid growing segment for instrumentation
•Cheaper ?•Potential for specific extraction
Cons•Manipulation of small volume samples
•For small to medium quantity of DNA•Magnetic stand/plate needed•Usually generic extraction
•Not all procedures equal in regards of time requirement
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NA extraction by hybridization
Different companies have developed over the past years, specific hybridization capture procedures:
RNAture (Hitachi Chemical), Xtrana, ProLinx, Gen-Probe, DNA Research Innovation (DRI now part of Invitrogen/Life Technologies) have been the most visible.
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Gen-Probe Target Capture Technology
Target Capture•Maximizes assay sensitivity & specificity. •RNA target is “captured” with magnetic beads.•Eliminates inhibitors in swab and urine samples.
•Allows for simplified specimen processing.•Patented process with specific probes coated magnetic particles.
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How it works ChargeSwitch Technology provides a switchable surface charge that is dependent on the pH of surrounding buffer to purify nucleic acids (Figure 1). In conditions of pH < 6.5, the CST surface has a positive charge that selectively binds the negatively charged nucleic acid backbone. Proteins and other contaminants are removed in an aqueous wash buffer. For elution, the charge on the CST surface is switched off by raising the pH to 8.5. Purified nucleic acid elutes instantly and is ready for downstream use.
Advantages of the ChargeSwitch Technology • Novel, pH dependent switch action • Avoids introduction of ethanol, organic solvents or chaotropic salts • Improved downstream performance for high specification applications • Unmatched high sensitivity for micro samples
Invitrogen ChargeSwitch Technology
ChargeSwitch gDNA50-100 µl
Lysis 1 min RTCapture 1 min
Washing 3x1 minElution 1+4 min
Total @ RT 10 min
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Very recent new approach for NA extraction
Enzyme based NA extraction
DNA extraction using a thermostable proteinase.…hands off, closed tube and all you need is a thermal cycler.
WorkFlow
automated on the epMotion 5075MC
Zygem: PrepGEMand ForensicGEM
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Very recent new approach for NA extraction
Boreal Genomics instruments are based on SCODA: a novel, proprietary electrophoresis technology that selectively concentrates nucleic acids from large sample volumes to a small, concentrated amount of gel or buffer. Advantages:
•Up to 100X more efficient for contaminant rejection•High yield even in very dilute samples•Simple operation•Target length selection during extraction•Protection of high molecular weight targets•Potential for sequence specific extraction including short RNA/DNA fragments
This is accomplished by means of rotating electric fields that act uniquely on long, charged polymers and leave other molecules unaffected.
1x5mL or 4x1 mL input
• Target run times < 30 min
• Digital high voltage control
• Unattended operation
• Lysate to purified sample in one step
• Extraction in 10 – 50 uL of buffer
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NA extraction Automation: why?
Time consuming step in MDxSample throughput
Hand on timeReproducibility
StandardizationCost
Instrumentation availability
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NA extraction automation options
✓Microtiter Plate based Procedures
✓Generic Robotic Instrumentation
✓Dedicated Robotic Instrumentation
✓In Vitro Diagnostic Robotic Instrumentation
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Microtiterplate based NA extraction
•Qiagen and Macherey-Nagel have been pioneers in providing DNA extraction protocols on 96 well-plates
Qiagen, http://www.qiagen.com
DNAeasy, RNAeasy, QIAamp, QIAfilter
Macherey-Nagel, http://www.macherey-nagel.com
NucleoBond, NucleoSpin, NucleoTrap, Multi-96
•Additional companies providing 96 well-plate compatible kits for DNA/RNA extraction:
Promega, BioLine, Stratagene, Millipore, Ambion, Invitrogen…
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High Throughput DNA extraction with Macherey-Nagel
The NucleoSpin Multi96 blood kit from macherey-Nagel allows rapid parallel DNA extraction from blood, serum, plasma, cell cultures, buffy coats.
Features:
silica membrane technology
complete removal of PCR inhibitors
no organic solvents
200 µl whole blood or 107 lympho. or cultured cells can be processed
Simultaneous processing of 192 samples
2 x 96 well plates
or
n x 8 strips on a 96 deep-well block
Processing time >2hrs
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Microtiterplate compatible viral DNA extraction
Bench top centrifuge for Nucleospin multi 96
DNA extraction.
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Semi-Automation of the QIAamp 96 Spin Blood Kit on the Biomek 2000
Setup of the Beckman Coulter Biomek 2000
Tablet
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Centrifugation of the Filtration Unit + Disposable Deep-Well
Semi-Automation of the QIAamp 96 Spin Blood Kit on the Biomek 2000
Centrifugation of the Filtration Unit + Storage Deep-Well
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NA extraction automation options
✓Microtiter Plate based Procedures
✓Generic Robotic Instrumentation
✓Dedicated Robotic Instrumentation
✓In Vitro Diagnostic Robotic Instrumentation
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Generic Robotic Instrumentation
✓Develop your own procedure
based on manual protocols
some are robotic friendly
✓Follow reagents providers recommendations
✓Follow robotic instrument providers recommendations
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Generic Robotic Workstations with available molecular testing applications.
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Company Instrument IVD-D or CE-IVD compliant; comments
NA extraction applications
available
PCR applications available
Web Address
Agilent Automation Solutions
Biocell, Bravo, BenchCel
No; very high throughput Yes Yes chem.agilent.com/
Aurora Instruments Versa serie, Versa mini no Yes Yes aurora-instr.com/
Beckman Coulter, Inc. Biomek 2000,3000Biomek NX,FX No Yes Yes beckmancoulter.com/Beckman Coulter, Inc. Biomek 2000,3000Biomek NX,FX No Yes Yes beckmancoulter.com/
Caliper LifeSciences
SciClone ALH3000 No Yes Yes
caliperls.com/Caliper LifeSciences
SciClone ALH3000 No Yes Yes
caliperls.com/Caliper LifeSciences Zephyr No Yes Yes caliperls.com/Caliper LifeSciences
Twister II+AB7900 No No Yes
caliperls.com/
Cybio CyBi-Dilus, -RoboSpense No Yes Yes cybio-ag.com/
EppendorfepMotion 5070
No; MC with integrated PCR Yes Yes eppendorf.com/Eppendorf epMotion 5075
LH, VAC, MC, TMX
No; MC with integrated PCR Yes Yes eppendorf.com/
Hamilton STAR, STARlet, Nimbus
Yes; NAT pooling system Yes Yes hamiltoncomp.com/
Labcorp-Protedyne BioCube, Radius No; very high throughput Yes Yes protedyne.com/
Perkin-Elmer Janus CS Autoplex, Forensic No Yes Yes las.perkinelmer.com/
Tecan Freedom Evo Yes Yes Yes tecan.com/
ThermoCRS CRS DNA No; very high throughput Yes Yes thermo.com/
Xiril Xiril 75, 100, 150 No Yes Yes xiril.com/
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ThermoCRS Extraction platforms
High throughput (up to 14, 96-well microplates/hour)Proven reliabilityUses well-established DNA purification protocolsAdaptable to any magnetic bead basedseparation methodUnattended 24 hour operation and remote monitoringPOLARA™ Open Architecture Laboratory Automation Software
The CRS DNA Purification System is an automation tool for beadbased methods using magnetic particles. This includes binding of DNA
to magnetic beads, washing of the bound material, elution from the solidphase and transfer to a final destination plate.
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Agilent Automation Solutions: Bravo and BenchCel
Bravo
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A flexible workstation providing high-throughput gDNA isolation
• Up to 10 microplates can be processed in one run without user intervention using the Invitrogen Charge Switch EasyPlex procedure• Microplate processing time is approximately 10 min per microplate (excluding incubation time ~ 30min)
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In May 2007, Protedyne became a wholly owned subsidiary of Laboratory Corporation of America.
Protedyne BioCube Platforms
Protedyne BioCubes
LX, SX, EX
Performance: Approximately 7,000 DNA samples can be prepared per eight-hour shift using a Protedyne 96-well,
vacuum-based plasmid DNA purification procedure.
Also, QiaAmp Genomic DNA on the BioCube: 14 plates per 8
hour shift
Protedyne Radius
Protedynes bench-top robotic system
a protocol for the Invitrogen ChargeSwitch EasyPlex gDNA kit using the gDNA isolation workstation in which up to 10 microplates (96-well) can be processed simultaneously.
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Tecan Extraction Platforms
The MiniPrep , f rom Drug Discovery (mother - daughter plate replication), to Molecular Biology (Sequencing preparation, PCR a m p l i fi c a t i o n a s s a y s ) a n d Diagnostics (Immunophenotyping, ELISA Assays).
The Genesys serie with four or eight tip arm for high throughput. NA extraction automation has been fully automated using the magnetic separator MagS and solid phase extraction through columns. The extraction is usually performed in 96 well cartridge blocks. These are placed on vacuum chambers on the instrument worktable and can be evacuated by software controlled vacuum pump VacS.
The Freedom Evo family from 1m to 2m deck size. A special mention for the IVD-D compliant Freedom Evo Clinical.
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Xiril Robotic Platforms
X75 X100
Xiril Robotic Workstations have a 1-tip, 2-tip or 4-tip liquid handling arm with selectable and exchangeable pump volumes. Each pump is equipped with a tip adaptor that fits disposable tips with or without filters.
For added flexibility and increased sample throughput, the Xiril X100 and X150 can be configured with an additional liquid handling arm for specific Life Science applications. Macherey, Agencourt, Promega application notes available.
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Robotic Workstation Industrials “Partners”
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Beckman Coulter Hamilton Thermo-Jouan- CRS Robotics
PerkinElmer Tecan Self-Contained units
Biomek 2000, Biomek 3000 MicroLab, Starplus, Starlet
CRS DNA Purification System
MultiProbe IIEX, DNAtrack, Janus
Genesis RSP 100, 150, 200Evo
Agilent Biocel, Bravo, Protedyne BioCube
Biomek FX, Biomek NX AutoSprint MiniPrep 60 MolBio
Multimek
Promega, Xtrana, Agencourt, Qiagen
Millipore, Agowa, Roche BilaTec Agencourt, Agowa, Macherey-Nagel,
Promega
Qiagen, Macherey-Nagel, Agowa
Various, Invitrogen
Filtration, Magnetic, hybridation
Filtration, purification Magnetic Filtration, Magnetic Filtration, Magnetic all types
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Robotic friendly NA extraction reagents
๏ Automated Protocols
๏ Automated SV 96 Total RNA Isolation System Protocol
๏ Automated Wizard® Magnetic 96 DNA Plant System Protocol
๏ Automated Wizard® SV 96 Genomic DNA Purification System Protocol
๏ Automated Wizard® SV 96 PCR Clean-Up System Protocol
๏ Automated Wizard® MagneSil™ 384-Well PCR Clean-Up System Protocol
๏ Automated Wizard® MagneSil™ 384-Well Sequencing Reaction Clean-Up System Protocol
๏ Automated Wizard® MagneSil® 96-Well PCR Clean-Up System Protocol
๏ Automated Wizard® Magnetic 96 DNA Plant System: Scaled Up Protocol for Increased DNA Yield
๏ Automated Wizard® MagneSil® Sequencing Reaction Clean-Up System Protocol for 96-Well Plates
๏ Automated MagneSil® ONE, Fixed Yield Plant Genomic DNA Purification Protocol
๏ MagneSil® KF, Genomic System
Example of Promega’s reagents
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NA extraction automation options
✓Microtiter Plate based Procedures
✓Generic Robotic Instrumentation
✓Dedicated Robotic Instrumentation
✓In Vitro Diagnostic Robotic Instrumentation
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Dedicated Workstations For NA extraction 1/4
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Applied BioSystemsApplied BioSystems
AutogenAutogenAutogenAutogen
Beckman Coulter, Inc.Beckman Coulter, Inc.
Bee RoboticsBioer
BiomérieuxBiomérieux
Bioneer
ABI Prism 6100 Filtration 96 96/30min jusqu’à 1m de cellules
ARN, ADN appliedbiosystems.com
ABI Prism 6700 Filtration; archivage, préparation PCR
96 96/75min jusqu’à 1m de cellules
ARN, ADN
appliedbiosystems.com
AutoGenflex 3000 Centrifugation 48 48/4-6hr jusqu’à 5mL ADN, ARN, plasmides, cosmides
autogen.com
AutoGenPrep 2000
Centrifugation
48 48/4-6hr 1mL ADN, tissus, plasmides, cosmides, levures, plantes
autogen.com
AutoGenPrep 245
Centrifugation
24 24/3.5hr 700µL ARN, ADN, tissus, plasmides
autogen.com
AutoGenPrep 965
Centrifugation
4x96 384/4-6hr Jusqu’à 250µL ou 1mL
ADN, plasmides, cosmides
autogen.com
Vidiera NsP Filtration; quantification, normalisation, préparation PCR
96 96/2.5hr Jusqu’à 300µL ADN, ADN et ARN viral
beckman.com
SPRI-TE Magnétique/Magtration; cartouches de réactifs
10 10/30min NS ADN
beckman.com
QuatroProbe Magnétique 48 NS 200µL à 1mL NS beerobotics.com
Gene-Pure NPA-32 Magnétique/Magnetic rod
32 NS 50 à 800µl ADN, ARN bioer.com.cn
Extractor Méthode de Boom 10 120/8hr ADN 100µL, jusqu’à 2mL
ADN, ARN biomerieux.com
EasyMag Magnétique/Méthode de Boom
24 60min Jusqu’à 1mL
biomerieux.com
HT-Prep Pro Filtration 96 60min Jusqu’à 300µL ADN, ARN bioneer.co.kr
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Dedicated Workstations For NA extraction 2/4
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ChemagenChemagen
Corbett LS part of QiagenEppendorf
FujiFilmFujiFilmFujiFilm
GeneSystemsGenomic
HandyLab part of BD
Invitrogen
Magnetic Biosolutions AB
Mole
NordiagNordiag
Promega
Chemagic Magnétique/Magnetic rod
12, 96 150/8h 10mL (45min/12), 4000/8h 1.5mL (15min/96)
10µL à 10mL DNA, RNA, RNAv, mRNA
chemagen.de
Prepito
Magnétique/Magnetic rod
1-12 30min 10µL à 600µL
DNA, RNA, RNAv, mRNA
chemagen.de
QIAxtractor FIltration 8-96 8/45min, 96/96min 180 à 200µL DNA, RNA, RNAv, mRNA
corbettlifescience.com
epMotion 5075 VA, TMX
Filtration 1-96 NS NS DNA, RNA, tissus, plasmides
eppendorf.com
QuickGene-610 Filtration sur Membrane poreuse de 80µM
6 12min 2mL ADN fujifilm.com
QuickGene-810Filtration sur Membrane poreuse de 80µM
8 6min 200µl ADN, ARN, tissus, plasmides
QuickGene-mini80
Filtration sur Membrane poreuse de 80µM
8 6min (manuel) 200µl ADN, ARN, tissus, plasmides, cellules
GeneExtract Filtration 6 Semi-automatique > 1L de liquide ADN genesystems.fr
ExtraGene Centrifugation 48 240/jour 0.5 à 10mL ADN, ARN, Proteines genomics-tools.com
Lynx Magnétique; cartouches de réactifs
24 35min NS ADN, ARN handylab.com
iPrep Magnétique/Magtration; cartouches de réactifs
12 26min 350µL ADN invitrogen.com
Magnatrix 1200 Magnétique/Magnatrix 12-96 20min/12 NS ADN, ARN magbio.com
Magnatrix 8000plus
Magnétique/Magnatrix
8-192 NS NS
GeneMole Magnétique; cartouches de réactifs
16 NS 50µL à 1mL ADN, ARN, cellules, tissus
molegenetics.com
Arrow Magnetic, Bug’s and Beads
1-12 30min 200-500µL DNA, RNA nordiag.com
Bullet 1-96 1.5hr 20 à 200µL DNA, RNA
nordiag.com
Maxwell 16 Magnétique/Magnetic rod; cartouches de réactifs
16 30min 50 à 400µL ADN www.promega.com
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Dedicated Workstations For NA extraction 3/4
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Precision System SciencePrecision System SciencePrecision System SciencePrecision System SciencePrecision System SciencePrecision System Science
QiagenQiagenQiagenQiagenQiagenQiagenQiagen
Mag. Syst. 6Mx Magnétique/Magtration; cartouches de réactifs
6-48 NS 100 à 1000µl ADN, ARN pssbio.com
Mag. Syst. 8Lx Magnétique/Magtration; cartouches de réactifs
1-8 50min 1-10mL ADN
pssbio.com
Magt. Syst.12GC Magnétique/Magtration; cartouches de réactifs
1-12 30min 100 à 200µL ADN, ARN, ADN et ARN viral, tissus, plasmides
pssbio.com
SX-6G Magnétique/Magtration 48 80min 25 à 500µL ADN, ARN
pssbio.com
SX-8G Magnétique/Magtration 96 80min 5 à 40µL ADN, ARN
pssbio.com
SX-96GC Magnétique/Magtration 4x96 NS 10 à 40µL ADN, ARN
pssbio.com
Mag. Syst.Ba-X Magnétique/Magtration 1-3 0.2ml,1ml, 3ml ADN, ARN
BioRobot Universal SystemFiltration + centrifugation + Magnétique; PCR setup
4x96 4x96/2-3hr jusqu’à 200µL ADN, ADN et ARN viral, plasmides
qiagen.com
BioRobot MDX DSP** Filtration + centrifugation + Magnétique ; PCR setup; CE IVD-D
4x96 4x96/2-3hr jusqu’à 200µL ADN, ADN et ARN viral
qiagen.com
EZ1, EZ1 advanced, EZ1 advanced XL
Magnétique/Magtration; cartouches de réactifs; CE-IVD-D
6, 6, 14 20min 200 à 350µL ADN, ADN et ARN viral, tissus
qiagen.com
BioRobot 48/96 Magnétique/Magtration 0,5 48/2hr 100 à 300µL ADN, ADN/ARN, ARN, mARN
qiagen.com
BioSprint 15/96 Magnétique/Magnetic rod
0,15625 20min 10µL à 1mL ADN, ADN/ARN, ARN, mARN
qiagen.com
AutoPure LS Centrifugation 16 96/jour Jusqu’à 10mL ADN
qiagen.com
QIACube Filtration + centrifugation
12 65min jusqu’à 200µL ADN, ADN et ARN viral, plasmid
qiagen.com
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Qiagen QIAsymphonySP, RGQ Magnetic, Magnetic rod 300 LBC/16hrs; 1-96;
1-72 2hrs up to 1ml DNA, RNA qiagen.com/
Dedicated Workstations For NA extraction 4/4
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Roche DiagnosticsRoche DiagnosticsRoche Diagnostics
Thermo LabSystemsThermo LabSystems
MagNAPure 2.0/96 Magnetic, Magtration; PCR prep
32/96 2hr/1hr 20 à 300µL DNA, RNA, DNA/RNA, mRNA
roche-applied-science.com
MagNAPure Compact SystemMagnetic, Magtration; cartridge
8 20min 100µL à 1mL
DNA, RNA, DNA/RNA, mRNA
roche-applied-science.com
AmpliPrep Magnetic,PCR prep, CE IVD-D, continuous flow
1-72 24/1hr 100 à 750µL DNA, RNAv
roche-applied-science.com
KingFisher Magnetic,Magnetic rod 2x12 24/17min, 29min, 41min
20 à 200µL DNA, RNA, mRNA, proteins, cells
thermo.com/
KingFisher ML/96
Magnetic,Magnetic rod
15-96 15/14min, 36min, 43min
10µL à 1mL
DNA, RNA, mRNA, proteins, cells
thermo.com/
QIAEnsembleSP Magnetic, Magnetic rod 3000 LBC 16hrs
LBC, Urine Vials, PreservCyt, surepath Vials, ETU tubes
DNA qiagen.com/
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A closed system for the process of 8 samples / 4 hrs.
A precursor in dedicated workstation
The ABI 340 extractor
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AutoGen Centrifugation based Workstations
AutoGen extractors
AutoGenFlex Star
AutoGen 960-965 (4x96 samples in 4hrs)
Gene Prep
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Autogen - 960-965 Extractors
1. Processing Method: • Single phase organic extraction2. Protocols: DNA extraction from whole blood, blood spots, human tissues and cells (including ES cells), plasmids, BACs, microbes and plants. 3. Sample Volume:• Up to 1.8 mls per well4. Maximum Sample Throughput:• 384 (96 deep well plate x 4) per run5. Processing Time:• Four hours for 4 plates
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AutoGen - AutoGenFlex StarAn AutoGen and QIAGEN Collaboration
High precision gripper-type robot hand
Transports sample tubes to and from workstation, dispensing station and centrifuge
Removable sample and DNA storage racks
Heater to allow for efficient digesting of the sample and drying of purified DNA
-4-position swing type buckets-Variable speed and duration settings-HEPA-Filter for biohazard reduction
A u t o G e n F l e x i s c o m p l e t e l y automated and easy to use, with no manual intervention required. Isolate up to 80 x 10ml whole blood samples per day
58Qiagen's proven FlexiGene chemistry
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AutoGen - Gene PREP
•Automates entire extraction process including on board digestion.•Capable of extraction from multiple sample types.•Low cost per prep using AutoGen’s reliable solution phase extraction chemistry.•Higher throughput based on new design which shortens processing time.•Produces high quality and high yields of DNA.•Optional tube stacker allows for unattended processing of up to 192 samples.
48 samples in 2 hours (faster time to result).Direct application of extracted nucleic acid to your next procedure (saves labor and time).
The GENE PREP is the latest fully automated DNA extraction system from AutoGen.
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On-Board Centrifuge and XYZ Robot
Heating ShakerOptional Tube Unit Stacker
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Genomic ExtraGene Platform
ExtraGene is a multi-protocol platform based on centrifugation.
ExtraGene process up to 48 samples in 20 ml tubes. The running time depends on the protocol.
Up to two improved centrifuges, with a mechanized hatch on the top
The robotic arm moves a special tool for tubes cap opening, closing, tube handling, nozzles for syringe pumps and precision peristaltic pumps
On the front side, a drawer supports tubes racks, tips racks and liquid and solid waste collector
ExtraGene is contained in a fume-hod (optional)
Generic reagents = Much more extractions with your budget
Full automation = Much more extractions with your work force
Separation by centrifugation = Higher yeld and lower cost (less than 1.5€ / extraction)
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genomics-tools.com
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Filtration based platform on a centrifuge
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The QIAcube process QIAGEN spin columns, enabling seamless integration of automated, low-throughput sample prep into your laboratory workflow. No change of purification chemistry is required, assuring fast startup and immediate results. All steps in the purification procedure are fully automated – including sample lysis – and up to 12 samples can be processed per run.
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Beckman Coulter Inc. Vidiera NsP
Primary tubes on racks
NA extraction by filtration
Qiagen chemistry
Quantification by OD
Normalization
PCR setup
Archieving
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“High throughput” nucleic acid automated extraction with minimum hands-on-time and a very fast turnaround time (40 minutes for 24 extractions).
Biomerieux Nuclisens EasyMag
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PSS Workstations-new concept
Individual barcoding for reaction cartridges
CCD camera procedure monitoring
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PSS Workstations-new concept
Large volume magnetic extraction- PSS-8LX
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0.2 to 3 ml sample volume handling
1 to 10 ml sample volume
50 min run
pre-filled reaction cartridge
The new Magtration System BA-X
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Beckman Coulter Inc. SPRI-TE
Automated processing of 1-10 samples in 30 minutes
✓ High-performance SPRI magnetic bead technology
✓ Pre-packaged, ready-to-use reagents and labware
✓ Simple to operate✓ Method card driven protocols✓ UV decontamination
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Nordiag Magnetic Extraction
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The affordable NorDiag Arrow instrument makes automated sample preparation possible for a l l l a bo ra t o r i e s . The new innovative tip pump assembly will allows for a wide range of sample volumes for extraction. The tip pump assembly is a closed system, reducing the possibility for contamination. Touch screen operation which makes the instrument very user friendly.
The NorDiag Bullet, 8 tip config.
96 samples from primary tube to PCR*/SDA** setup in less than 1.5 hours, using the NorDiag Bullet instrument
The BUGS’n BEADS technology is based on an initial adsorption of bacteria/cells to magnetic particles, followed by a magnetic separation where the sample mater ia l i s removed. This step removes most of the inhibitors that are often found in samples. The next step is lysis of cells and the release of nucleic acids, followed by immobilisation of nucleic acids to magnetic particles.
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Qiagen Magnetic Extraction
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EZ1 Advanced
BioRobot M48 workstation BioSprint 96 Workstation QIAsymphony Workstation
EZ1 Advanced XL
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2010 Evolution in Roche Automated Platforms
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The Magna Pure 96 System
Speed - 96 sample extraction in <1hrEasy loadingPrefilled reagent traysFewer disposablesRun surveillanceConnectivity
Time from sample to results: 2 hrs
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Chemagic by Chemagen
96 rods for <200 µl samples in 96 well plates
4000 samples/day (200µl/6µg)
12 rods for <10 ml samples in 15 or 50 ml tubes
12 samples/40 min (7ml/210µg)
Distributed in the US by Parallabs86
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Chemagic by Chemagen
1 – 12 samples per run๏ integrated buffer dispensing๏ revolutionary resuspension technology๏ standard plastic devices๏ chemagic Kits and protocols for a huge variety of
sample materials๏ quantitative yield, highest purity๏ sample sizes up to 600 µl๏ bar code reading available
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NA extraction automation options
✓Microtiter Plate based Procedures
✓Generic Robotic Instrumentation
✓Dedicated Robotic Instrumentation
✓In Vitro Diagnostic Robotic Instrumentation
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Abbott Molecular solutions
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m2000sp m2000rt
RealTime HIV-1
RealTime HCV
RealTime HCV Genotype II
RealTime HBV
RealTime High Risk HPV
RealTime CT/NG
RealTime CT
RealTime mS9
Parvo B19 PCR Kit
HSV 1/2 PCR Kit
CMV PCR Kit
VZV PCR Kit
EBV PCR Kit
M. tuberculosis
SARS RT-PCR Kit
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Abbott Molecular solutions
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Abbott M24sp (Freedom EVO Clinical 75)
1 to 24 sample batch processingNA extraction, Open ModeNo PCR setupCE-marked
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Biomerieux NucliSens EasyMag Platform
“High throughput” nucleic acid automated extraction with minimum hands-on-time and a very fast turnaround time (40
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Qiagen platforms: New Generation
The QIAsymphony SP
•from 1 to 96 samples in batches of 24
•volumes up to 1 ml
•Magnetic Rod separation
•in-process sample loading
•bar code reading
•expandable
942008 2010 2011?
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Qiagen QIAsymphony RGQ
The QIAsymphony RGQ:
from DNA extraction with the QSsp, to PCR setup with the QAas and PCR with the Rotor-Gene Q (1 to 72 samples per run)
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HIV, HCV, HBV and CMV, EBV, HSV, VZV, BKV1 Artus RealTime PCR kits available for the Rotor-Gene Q. More to come.
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Roche Cobas p630 System
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The cobas p 630 Instrument provides fully automated pre-analytical solution for primary tube handling.
It can be used in conjunction with the COBAS® AmpliPrep/TaqMan® System and can support up to three COBAS® AmpliPrep Instruments.
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Roche Cobas 4800 System
Streamlined workflow
• Nucleic acid extraction from multiple types of specimens is fully automated, with the fewest manual steps• Swab and urine samples in cobas PCR Media in asingle run • Multiple liquid cytology media types in a single run • No manual preprocessing• Cobas PCR Media tubes and liquid cytology vials can be loaded directly into the system• Load-and-Go reagents are already bar-coded and ready to use, requiring no additional mixing• Bidirectional connectivity to the LIS enables the automated reporting of results
Flexibility to meet different needs• Adapts to the needs and throughput of each lab• Can run up to 376 samples in a day but is still efficient for labs with less intensive testing needs
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Siemens Medical Solutions Diagnostics
Primary tube sampling
Up to 96 samples per run
Proprietary extraction technology using mag. particles
Siemens
Competition
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From sample Prep Automation to ? with the BD Viper
The BD Viper System is a patented approach to laboratory automation using an industrial class of robotics known as Selective Compliance Assembly Robot Arm (SCARA).
This classification indicates that the robot is multi-jointed (4-axis), similar to the human arm.
The BD Viper Sample Processor automates the sample hand l ing associated with high volume amplified molecular tes t ing of Chlamydia trachomatis and Neisseria gonorrhoeae performed on the BD ProbeTec ET System.
More than 500 samples for CT and GC can be processed per 8-hour shift.
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The BD Viper with XTR Technology
ThroughputUp to 736 CT/CG results in 8.5 hour shift
Time to First Results3 hours 15 minutes
Amplification and Detection TechnologyStrand displacement amplification with real-time amplification and detection
Contamination PreventionClosed solid barrier amplification system
Specimen Collection TypeVaginal & endocervical swabs, urethral swabs, urine, liquid-base cytology (LBC). All collection devices utilize pierceable caps during processing
On-Board Sample Capacity96 samples including controls per run
Patient Sample Identification LocationA membrane switch pad records the location of the tube. Capac i ty to read mult ip le barcode configurations: 2 of 5, code 39, code 128, codabar
Sample Dispensing System6-station pipettor with liquid-level sensing
Sample Volume800 µL
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I wish I could
see the XTR
• On-board sample extraction utilizing proprietary BD Fox™ chemistry
• Walk-away platform with "load and go" processing• Automation using SCARA robotic technology• Multiple sample types – Liquid-based cytology,
male urethral swabs, endocervical swabs, vaginal swabs, and urines
• Zero reagent preparation – Ready-to-use reagents• Automated sample cap piercing• Liquid-level sensing of samples and reagents• Patient sample location identification
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Gen-Probe DTS 800 Systems
DTS® 800 SystemsGenerates up to 800 results in 8 hours
Includes the primary equipment and ancillary items to process APTIMA assays
DTS: Simplified Sample ProcessingThe DTS family of systems uses an exclusive penetrable cap to minimize contamination and results in safer, more convenient sample removal.
The design also reduces the time and effort required to remove the cap and obtain swab samples from the tube.
DTS simplifies sample transport, minimizes handling and greatly reduces laboratory cross-contamination.・In addition, bar code
technology simplifies the preparation of worklists.
Sample Preparation SystemThe Ten-Tube Unit (TTU) design
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The Very 1st Fully Integrated MDx Platform
Complete automation of extraction, amplification and detection
one operator can test 500 samples with the APTIMA Combo 2 assay in approximately 8.5 hours, or 1,000 samples in approximately 13.5 hours. In addition, one operator can run two TIGRIS systems
simultaneously.
APTIMA Combo 2 assay for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC)
Procleix HIV-1/HCV Assay for simultaneously detection of HIV-1 and HCV
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Gen-Probe® Panther New System
1 the PANTHER system can continue running unattended, processing 500 samples in 12 hours.
2 Primary tube sampling with random access loading for maximum flexibility and productivity.
3 Multiple assays can be run from a single sample, and operators have continuous access to samples, reagents and consumables.
4 A customer-driven design, intuitive software for ease of use, and remote diagnost ics capabilities.
5 Extensive process controls that include positive sample identification, liquid level detection, reagent dispense verification, and radio frequency identification (RFID) tags on fluid containers.
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In Europe, the PANTHER system can initially be used to perform Gen-Probe's nucleic acid tests for Chlamydia and gonorrhea. These include the APTIMA Combo 2(R), APTIMA(R) CT (Chlamydia) and APTIMA GC (gonorrhea) assays.
Random accessOptimized workflow and throughputSTAT test prioritization Multiple tests from a single sample
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Fully Automated MDX Platforms EVolution
IVD industry Challengers:
Cepheid GeneXpert -> Cepheid Infinity
BD MAX System
Icubate, Idaho, Iquum, GeneFluidics
Upcoming or in Dev.
Hitachi High-Tech Open Platform
BCI UniCel DxN
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What Future for NA Extraction in MDx?
✓Automation
✓Magnetic beads = most popular✓Pre-dispensed reagents
✓Size of batch determination not final (6, 8, 24, 32, 48, 72, 96)✓96 sample batch mainly found on “generic” robotics
✓Semi-random and random access platforms start to be popular
✓Evolution through complete integration of NA extraction + PCR + Detection
✓More questions unsolved about sample preservation
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Freedom EVO / REMP SSS Factory
The Freedom EVO® and the REMP (Small Size Store™ (SSS) together enable the storage, retrieval and reformatting of compounds and DNA samples.
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Biophile ultra-low temperature automated
Biophile (part of TekCel)
a combination of freezer, automation and information technologies.
BIOPHILE, Inc., is the first to market -80C ultra-low temperature automated storage and retrieval systems.
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High Throughput Clinical Sample StorageARUP implemented the original Automated Transport and Sorting System built by MDS AutoLab of Toronto, Ontario on November 17, 1998. This system served ARUP's needs extremely well for more than five years, reaching a level of 18,000-22,000 new patient specimens per day and a total volume of tubes transported and sorted each day of 27,000 to 32,000.
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Sample Storage at room Temperature
DNA storage at room temperature instead of -20 or -80°C
๏Biomatrica, San Diego, CASampleMatrix is a dissolvable compound that stabilizes DNA at room temperature
SampleMatrix forms a protective seal around the sample as it dries, effectively ‘shrink-wrapping’ the DNA.
๏Genevault, Carlsbad, CAFTA paper on plate: GenPlates or GenTegra: Inorganic matrix
Outsourcing sample storage
๏Imagene, Bordeaux/Paris, FranceEncapsulating DNA, RNA or proteins in an aluminium-capsule under zero atmosphere
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Sample Storage at room Temperature
Biomatrica Preserve and store DNA at room temperature
Biomatrica has developed SampleMatrix, to stabilize and protect biological materials at room temperature without degradation. The SampleMatrix technology was designed by combining extremophile biology that enables long-term survival of organisms in extremely dry environments with synthetic chemistry.
Samples are preserved through formation of a protective thermo-stable barrier during a simple air-drying process. Samples are protected from degradation from heat and UV light.
DNAstable enables you to store plasmid and genomic DNA samples at room temperature. When you are ready to use your samples, 100% of the DNA can be recovered in 10 minutes or less and then used directly in downstream applications. By exploiting these unique characteristics, Biomatrica products enable researchers to stabilize biological samples for long periods of time.
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Sample Storage at room Temperature
GenVaultGenPlates’ proprietary format features FTA paper elements built into each wells for transport and long term storage of biosamples at ambient temperature, in the dry-state.
Upon application of 10ul of biosample directly to the 6mm disc of FTA paper, cells lyse and release DNA. The DNA becomes entwined in the fibrous network of the cellulose matrix and is dried completely at ambient temperature.
When DNA is required for downstream analysis, high-quality double-stranded DNA from FTA paper can be reliably recovered using the GenSolve chemistry.
Samples stabilized and dried down with GenTegra and stored for over 12 months at ambient laboratory temperature (21°C to 25°C) perform identically to DNA stored at -20°C in all downstream applications. Long-term accelerated stability studies of DNA at elevated temperature (76°C) indicate that DNA stabilized with GenTegra will retain its molecular integrity for decades at ambient laboratory temperatures.
Ensuring the dryness of DNA samples prior to storage is an important factor in preserving DNA integrity in ambient temperature storage conditions.
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Sample Storage at room Temperature
Imagene Room temperature DNA preservation
This technology relies in a DNAshell capsule which utilizes encapsulation under a controlled anhydrous and anoxic atmosphere of preliminarily desicated and purified DNA, into small sealed, corrosion-proof metallic capsules. This process prevents DNA from being altered by any degradation factors (water, oxygen, light).
Completely anhydrous, anoxic and perfectly airtight, the DNAshell capsules provides decisive advantages compared to freezing, enhancing stability, safety, transport and distribution of DNA. It also lowers operation and maintenance costs. In addition, the laser-marked Data Matrix codes on the capsules enable easy traceability and inventory of each DNA sample.
Ensuring the dryness of DNA samples prior to storage is an important factor in preserving DNA integrity in ambient temperature storage conditions.
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DNA can therefore be preserved at room temperature for unlimited periods.
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GenVault’s Dynamic Archive is an automated system for archiving and retrieving G e n P l a t e s a t r o o m temperature. The system utilizes a robot to access the shuttles that contain trays on which GenPlates are housed. Up to 31,500 geneplates.
GenVault’s DeskTop Archive is the smallest in the series of room-temperature biosample archiving systems available from GenVault. The “desktop” size system allows one to test-drive the concept of b i o s a m p l e s t o r a g e a t roomtemperature at low cost and low risk. Up to 1200 biosamples.
Robotics Archive or Personal Archive
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Biomatrica's dry storage cabinets employ passive control devices to ensure a stable humidity environment and are useful for laboratories where moisture levels or fluctuation is an issue. The cabinets can maintain relative humidity at <40% for the optimal room temperature storage of samples stabilized in Biomatrica’s products. Dry storage cabinets require no energy input and help your lab save money and "go green."
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Sample Management Softwares
Biomatrica
Greiner
Central Labo Europehttp://www.central-labo-europe.com/
http://www.biomatrica.com/
http://www.greinerbioone.com/
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And also: Genvault, GenoLogics...etc.
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