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GeneJET Plasmid Miniprep Kit Thermo Scientific™

Thermo Scientific GeneJET Plasmid Miniprep Kit utilizes an exclusive silica-based membrane technology in the form of a convenient spin column. The kit recovers up to 20 µg of high copy plasmid DNA per isolation procedure.

Highlights

Efficient—high yields of up to 20 µg of high quality plasmid DNA
Fast—procedure takes less than 14 minutes
Convenient—no phenol/chloroform extraction or alcohol precipitation required
Pure—purified DNA is immediately ready to use

Applications

• Fast isolation of high purity plasmid DNA suitable for all conventional molecular biology procedures, including:
• FastDigest or conventional restriction digestion
• Automated fluorescent and radioactive sequencing
• PCR
In vitro transcription
• Transformation

Principle

A bacterial culture is harvested and lysed. The lysate is then cleared by centrifugation and applied on the silica column to selectively bind DNA molecules at a high salt concentration. The adsorbed DNA is washed to remove contaminants, and the pure plasmid DNA is eluted in a small volume of elution buffer or water. The purified DNA is ready for immediate use in all molecular biology procedures, such as fast and conventional digestion with restriction enzymes, PCR, in vitro transcription, transformation, and automated sequencing. Components Resuspension Solution Lysis Solution Neutralization Solution Wash Solution (concentrated) RNase A Solution Elution Buffer Spin Columns Collection Tubes Detailed Protocol

Related Products
RNase A
Elution Buffer for GeneJET Plasmid Miniprep Kit
Lysis Solution for GeneJET Plasmid Miniprep Kit
Resuspension Solution for GeneJET Plasmid Miniprep Kit
Neutralization Solution for GeneJET Plasmid Miniprep Kit
Wash Solution for GeneJET Plasmid Miniprep Kit
Binding Buffer for GeneJET Plasmid Miniprep Kit

QuantiGene™ Homogenizing Solution Invitrogen™

QuantiGene Homogenizing Solution is used to prepare tissue homogenates from fresh, frozen, or formalin-fixed paraffin-embedded (FFPE) tissues. Homogenization of tissues using QuantiGene Homogenizing Solution releases RNA for detection and quantitation with QuantiGene assays. The homogenizing solution is one of two components in the QuantiGene Sample Processing Kit for tissues, and is used to supplement this kit when extra volume is needed.

Extra MagMAX FFPE DNA/RNA Ultra Reagents Applied Biosystems™

This package includes supplemental reagents for use with the MagMAX FFPE DNA/RNA Ultra Kit (Cat. No. A31881). These additional reagents consist of protease digestion buffer, binding solution, and DNA wash buffer and are specifically for use when processing >40 um sections. See the User Guide for details.

Bead Tubes for PureLink™ Microbiome DNA Purification Kit Invitrogen™

These are the same bead tubes that are included in the PureLink™ Microbiome DNA Purification Kit (Cat. No. A29790), offered separately for additional applications.

Genomic DNA Purification Kit Thermo Scientific™

Thermo Scientific Genomic DNA Purification Kit is a simple and rapid system for high quality genomic DNA purification from various sources including: whole blood, serum, cell lines, bacterial cells, plant, and mammalian tissues.

The kit is based on selective detergent-mediated DNA precipitation from crude lysate. The entire procedure is rapid only 20 to 25 minutes with a typical yield of 2 to 10 µg genomic DNA from 0.2 mL of blood.

High molecular weight genomic DNA purified with the kit is suitable for direct use in all common molecular biology applications: PCR, FastDigest and conventional restriction digestion, cloning, DNA sequencing, and Southern blot analysis.

Highlights

Efficient—2 to 10 µg of genomic DNA from 0.2 mL of blood
Fast—procedure takes approximately 20 minutes
Universal—purifies genomic DNA from various sources
Flexible—easy to scale up and down
Safe—does not involve phenol extraction

Applications

• Purification of high-quality genomic DNA from:
• Whole blood or serum
• Cell culture
• Plant tissues
• Mammalian tissues
• Epithelium samples
• Bacteria Includes Lysis Solution Precipitation Solution NaCl Solution Detailed Protocol

PureLink™ Genomic Wash Buffer 2 Invitrogen™

The PureLink® Genomic DNA Mini Kit and PureLink® 96 Genomic DNA Kit enable high-yield, high-purity DNA extractions from a wide variety of sample types, including blood, tissues, cells, bacteria, swabs, and blood spots, in a familiar silica spin-column or semi-skirted 96-well plate format. Using the PureLink® Genomic DNA Kits, you will:
Obtain higher gDNA yields and purity - optimized spin-column membrane and buffer formulations
Save time and money - use with a large range of sample sizes (e.g., 100 µl to 1 ml blood); 96-well version does not require special equipment
Increase flexibility - one kit works with a variety of sample types

How it works
The PureLink® Genomic DNA Mini Kit protocol improves upon the easy, familiar spin-column methods currently in use (Figures 1 and 2). Columns are newly configured to bind and release DNA more efficiently. Samples are lysed in an optimized buffer formulated to enhance proteinase K activity and eliminate protein contamination. The chaotropic salt binding buffer allows the highest DNA binding of any column method. Powerful wash buffers remove all traces of protein and salt. DNA is eluted in a low-salt buffer to allow for pH stabilization of the DNA in storage.

For higher throughput, use the PureLink® 96 Genomic DNA Kit (Figure 3). In this 96-well version, sample processing is performed with either a vacuum manifold or centrifugation at only 2,250 g, allowing you more flexibility in centrifuge choice. The semi-skirted plate design is compatible with most vacuum manifolds on robotic workstations.

Higher yields and purity
With the PureLink® Genomic DNA Kits, youll get greater yields of higher-purity gDNA (determined by A260/A280 measurements) with all of your sample types - from bacteria to tissues to blood to cells (Figures 4-6). The PureLink® 96 Genomic DNA Kit outperforms competitor products in both DNA yield and purity for multiple sample types, including blood (Figure 7). A260/A280 and A260/A230 ratios of PureLink® gDNA are higher than those obtained with other kits, indicating no contamination carryover of protein or guanidine using the enhanced PureLink® Lysis and Wash Buffers (Figure 7).

Contents and Storage:
The PureLink® Genomic DNA Mini Kit includes PureLink® Genomic Spin Columns, Collection Tubes, Digestion Buffer, Lysis/Binding Buffer, Wash Buffers, Elution Buffer, Proteinase K, RNase A, and the manual. The PureLink® 96 Genomic DNA Kit includes PureLink® Genomic Binding Plates, Wash Plates, Deep Well Plates, Foil Tape, Digestion Buffer, Lysis/Binding Buffer, Wash Buffers, Elution Buffer, Proteinase K, RNase A, and the manual. Store at room temperature. Guaranteed stable for one year when properly stored.

PrepSEQ™ Nucleic Acid Extraction Kit for Food and Environmental Testing Applied Biosystems™

The PrepSEQ Nucleic Acid Extraction Kit produces high-quality bacterial DNA and viral RNA samples for PCR-based detection from a wide range of food and environmental samples. The quantity and quality of the nucleic acid can greatly impact the success of the sample analysis process and the overall quality of the final result. Food and environmental samples can be challenging to process because they often contain PCR inhibitors. For laboratories looking to achieve better real-time PCR results and improve overall efficiency, the PrepSEQ Nucleic Acid Extraction Kit meets these needs.

Automated Sample Preparation for PCR-Based Assays
• Extract high-quality bacterial DNA and viral RNA
• Process a wide range of food and environmental samples
• Fast time-to-results
• Prepare hundreds of samples per shift per instrument

High-Throughput Food and Environmental Sample Preparation
PCR-based bacterial assays have become an important tool in food safety and environmental testing. The Applied Biosystems PrepSEQ Nucleic Acid Extraction Kit prepares high-quality bacterial DNA and viral RNA from food and environmental samples for PCR-based testing using magnetic bead-based extraction. In combination with the automated MagMAX Express-96 Deep Well Magnetic Particle Processor or the KingFisher Flex Purification System with KingFisher 96 Deep-well Head, this optimized solution enables laboratories to prepare a wide range of sample types at rates of up to hundreds of samples per eight-hour shift.

Streamlined Workflow
An easy-to-use, easy-to-learn protocol simplifies integration of the PrepSEQ Nucleic Acid Extraction Kit into the laboratory workflow. The same protocol can be used on a wide range of sample types, and the MagMAX Express-96 instrument or the KingFisher Flex Purification System with KingFisher 96 Deep-well Head automates many of the workflow steps to reduce hands-on time.

A Complete Solution
The PrepSEQ Nucleic Acid Extraction Kit is part of a fully integrated, start-to-finish solution for food and environmental testing. The kit has been performance-verified for food and environmental testing with MicroSEQ Microbial Detection Kits, the Applied Biosystems 7500 Fast Real-Time PCR System, and RapidFinder Express Software.

GeneJET Plasmid Midiprep Kit Thermo Scientific™

Thermo Scientific GeneJET Plasmid Midiprep Kit, sufficient for 100 preps, is designed for large scale isolation of high quality plasmid DNA from recombinant E. coli cultures. The kit utilizes silica-based membrane technology in the form of convenient spin column. Each prep recovers up to 200 µg of high copy number plasmid DNA. The purified plasmid DNA can be used in a wide variety of molecular biology procedures, such as restriction endonuclease digestion, PCR, cloning, transformation, automated sequencing, in vitro transcription, and transfection of robust cell lines.

Highlights

Efficient—high yields up to 200 µg of high quality plasmid DNA from 50 mL of bacterial culture
High concentration of DNA purified—> 400 ng/ µL
Convenient—spin column format, centrifuge or vacuum protocols
Fast—procedure takes 60 minutes, considerably faster than using gravity flow columns

Applications

• Isolated plasmid DNA is suitable for all conventional molecular biology procedures, including:
FastDigest restriction enzyme digestion
• Automated fluorescent sequencing
• PCR
• Cloning
• Transformation
In vitro transcription
• Transfection of robust cell lines

ExoSAP-IT™ Express PCR Product Cleanup Reagent Applied Biosystems™

Key features of ExoSAP-IT™ Express reagent:
5 minute protocol — Fastest turnaround time to results
One-tube, one-step PCR cleanup — Add ExoSAP-IT Express directly to PCR product
Novel enzyme technology — Irreversibly inactivated in just one minute at 80°C
Conserve PCR samples — 100% recovery of PCR products, regardless of amplicon length
Eliminate spin columns or beads — Significantly decreases time and errors while increasing yield
Compatible — No interference in downstream applications
Scalable — Treat a wide range of volumes
Multiple formats — Single tube, 8 tube strip, and 96-well plate formats offer flexibility from manual pipetting to automated workflows
Go green — Generate less waste with a single-tube solution to PCR cleanup

Overview
It is necessary to remove excess primers and unincorporated nucleotides from the PCR reaction prior to sequencing.

An enzymatic cleanup method offers precision and speed over technologies such as spin columns or beads. ExoSAP-IT Express reagent consists of two hydrolytic enzymes, a modified Exonuclease I and Shrimp Alkaline Phosphatase (SAP), in a specially formulated buffer. It is used to remove excess primers and dNTPs from a PCR mixture with no interference in downstream applications. This unique, highly stable, one tube solution allows for greater confidence and superior sequencing results (Fig. 1).

Innovative solution
Our scientists engineered Exonuclease I to have increased heat lability, allowing for inactivation in only 1 minute (Fig. 2). ExoSAP-IT Express reagent is added directly to the PCR product for a 4 minute cleanup at 37°C, followed by a 1 minute inactivation at 80°C, which provides accurate and consistent results in just 5 minutes.

Fastest PCR cleanup method
This novel technology allows for a significant reduction in sample cleanup time with minimal steps, providing the simplest workflow (Fig. 3). ExoSAP-IT Express reagent offers rapid turnaround times and improved efficiency of resources while delivering the quality that scientists have become accustomed to when using ExoSAP-IT for PCR product cleanup.

Conserve PCR samples — simple one step, 100% recovery
The ExoSAP-IT Express enzymatic cleanup method is designed to minimize errors by reducing your protocol to a single pipette step, allowing for automated or manual processing from a single tube or microtiter well. ExoSAP-IT Express reagent outperforms the competition by ensuring 100% recovery of all amplicon sizes, both short and long (Table 1).

ExoSAP-IT Express PCR Product Cleanup is active in commonly used PCR buffers, so no buffer exchange is required. After treatment, ExoSAP-IT Express reagent is inactivated by heating at 80°C for 1 minute. The treated PCR products are then ready for subsequent analysis in applications that require DNA to be free of excess primers and nucleotides.

MagMAX™ Cell-Free Total Nucleic Acid Isolation Kit Applied Biosystems™

The MagMAX Cell-Free Total Nucleic Acid Isolation Kit is designed for enrichment of all nucleic acid species present in cell-free biological samples such as plasma. The kit utilizes MagMAX magnetic-bead technology, ensuring reproducible recovery of high-quality nucleic acid that is suitable for a broad range of applications, including real-time PCR, digital PCR, and next-generation sequencing.

The magnetic bead–based purification format allows you to easily process a wide range of sample input volumes (1 mL to 6 mL). When used with the KingFisher Duo Prime or KingFisher Flex Magnetic Particle Processor, 6 to 24 plasma samples of 2-mL input volume can be isolated at once. Alternatively, samples can be processed manually with the use of a magnetic stand.

Features of the MagMAX Cell-Free Total Nucleic Acid Isolation Kit include:
• Automation-ready, phenol-free extraction
• Fast procedure allows 6 to 24 samples to be processed in 90 minutes or less when used with the KingFisher Duo Prime or KingFisher Flex Magnetic Particle Processor with a 24 Deep Well Head
• Flexible protocol accommodates sample volume inputs ranging from 1 mL to 6 mL of plasma
• No need for carrier RNA
• Elution volumes ranging from 15 µL to 60 µL
• Compatible with the Oncomine Lung cfTNA Research Assay

Go beyond cell-free DNA and get the total picture
Isolating and analyzing circulating cell-free total nucleic acid (cfTNA) from plasma and other cell-free samples serves as a non-invasive method to detect and monitor targets associated with certain disease states such as cancer.

The first step in attaining this valuable information is efficient nucleic acid isolation that specifically recovers the circulating nucleic acid species that contains information related to the disease state. The importance of cell-free DNA (cfDNA) is becoming established, but it may not tell the whole story. Cell-free RNA (cfRNA) sequences serve as critical biomarkers as well in the form of microRNAs and gene fusions. This inclusion of the cfRNA with the cfDNA provides an opportunity for researchers to expand their studies and advance human health.

The MagMAX Cell-Free Total Nucleic Acid kit is developed by the same Thermo Fisher Scientific scientists that brought you the MagMAX Cell-Free DNA Isolation Kit. The difference is the inclusion of all cfRNA species in the eluate, allowing you to increase the amount of information available from a single cell-free sample. Just like the cfDNA Isolation Kit, the cfTNA kit is designed for both manual and automated isolation of nucleic acid from a wide sample volume input range of 1 to 6 mL. The phenol-free, magnetic-bead format makes the kit fully amenable to automated isolation.

Thanks to the scalable format that allows for various sample inputs, it is possible to vary the number of samples processed with a single kit. For example, enough reagents are provided to process 50 samples of 2 mL plasma each. Alternatively, with the same kit it is possible to process 25 samples of 4 mL plasma each. Higher and lower input volumes are possible and the number of samples that can be processed will vary accordingly. This unique product attribute allows you to tailor the product to your specific needs.

Benefits of MagMAX magnetic bead–based purification
Magnetic beads offer many benefits compared to other technologies for isolating nucleic acid. Beads bind the DNA more efficiently than glass-fiber filters, resulting in higher and more consistent yields. Additionally, because filters and vacuum manifolds are not used, there is no risk of filter clogging due to cellular particulates in samples. This clogging issue is of particular concern with protein-rich, large-volume samples such as plasma that are commonly used in circulating cell-free nucleic acid studies.

resDNASEQ™ Quantitative E. coli DNA Kit Applied Biosystems™

This quantitative PCR (qPCR)-based kit—part of the resDNASEQ® E. coliResidual DNA Quantitation System—enables highly sensitive quantitation of host cell DNA from E. coliexpression systems, typically in less than 4 hours.

The kit is part of an integrated system that includes sample preparation, TaqMan® Assay and master mix, standard DNA, instrument, and software. The system overcomes the limitations of traditional methods by combining high-recovery PrepSEQ® sample preparation and TaqMan® Assay-based quantitation of residual DNA.

Highly sensitive quantitation using proven TaqMan® real-time qPCR technology (Figure 1)
• Manual and automated sample preparation, optimized for quantitative recovery from complex sample matrices (Table 1)
• Consistent performance across the expected range of DNA fragment sizes (Figure 2)
• Integrated system from sample to results with sample preparation, master mix, TaqMan® primer/probe mix, and genomic DNA standard


Rapid, Sensitive Detection of E.Coli DNA
The resDNASEQ® E. coli Residual DNA Quantitation System is a quantitative PCR (qPCR)-based system for the detection of host cell DNA fromE. coli, an expression system commonly used for the production of recombinant proteins. Reliable and rapid, the system enables sensitive (LOQ = 15 pg DNA/ml test sample, Figure 1) and specific (Figures 2 and 3) quantitation ofE. coli DNA, typically in less than 4 hours. This performance helps ensure a high degree of confidence in quantitation data obtained from a broad range of sample types—from in-process samples to bulk drug substances—whether a sample contains high molecular weight or sheared DNA (Figure 3).

Table 1. DNA recovery using manual PrepSEQ® sample preparation protocol.
Assay performance data using 10 pg E. coli genomic DNA spike per sample, 3 analysts, and 9 test samples.

50 mg/ml protein sample
Mean Recovery Mean %CV
E. coli 83%5.04%


For Research Use Only. Not for use in diagnostics procedures.

Silica Bead DNA Gel Extraction Kit Thermo Scientific™

Thermo Scientific Silica Bead DNA Gel Extraction Kit is a simple and efficient system for DNA extraction from agarose gels and reaction mixtures. The kit utilizes the modified protocol of Vogelstein and Gillespie, employing solubilization of the agarose gel and selective adsorption of nucleic acids on specially prepared silica particles at high salt concentration. Silica particles bound with DNA are washed to remove contaminants and pure DNA is eluted with TE buffer or water.

This method requires few manipulations, and is both faster and easier to perform than other organic-based extraction methods. Compared to column based gel-extraction kits, the DNA Gel Extraction Kit offers flexibility for scaling reaction and elution volumes up or down. The purified DNA is suitable for all common molecular biology procedures, including restriction digestion, cloning, sequencing, etc.

Highlights

Flexible—easy to scale up or down both reaction and elution volumes
Efficient—greater than 80% DNA recovery
Fast—purification takes only 15 to 20 minutes
Effective—suitable for DNA fragments as short as 100 bp
Safe—does not involve phenol extraction

Applications

• Purification of DNA fragments from agarose gels prepared with TAE or TBE buffers
• Concentration of DNA samples for desalting or buffer change
• Removal of enzymes after PCR, restriction digestion, dephosphorylation or labeling reactions
• Removal of unincorporated nucleotides, primers and primer-dimers
• Removal of linkers following ligation
• Removal of residual salts, phenol, chloroform, or ethidium bromide
• Purification of RNA-free plasmid DNA

Includes Silica Powder Suspension Binding Buffer Concentrated Washing Buffer TBE Conversion Buffer Detailed ProtocolNote

Care should be taken to avoid DNA damage with UV light when the gel-purified DNA fragment will be used in downstream cloning reactions. Always use a long wavelength UV (360 nm) light-box during excision of the gel slice. If only a short-wavelength UV (254 to 312 nm) light-box is available, minimize the UV exposure to a few seconds or keep the gel on a glass or plastic plate. Alternatively, visible dyes can be included in standard agarose gels to allow for visualization of the DNA bands in ambient light (2, 3).

PureLink™ Expi Endotoxin-Free Mega Plasmid Purification Kit Invitrogen™

The PureLink Expi Endotoxin-Free Mega Plasmid Purification Kit enables quick and simple isolation of endotoxin-free plasmid DNA using an enhanced anion exchange membrane. In as little as 2 hours, purify up to 5 mg of high-quality plasmid DNA that is suitable for advanced transfection applications such as transfection of sensitive cells lines or genome engineering.

Advantages of the PureLink Expi Endotoxin-Free Mega Plasmid Purification Kit include:
• Simple and fast protocol—purification typically takes 2 hours
• High yield—isolate up to 5 mg of high quality plasmid DNA
• Endotoxin-free plasmid—<0.1 EU/µg, ideal for sensitive applications
• High purity—ultralow levels of protein, RNA, and genomic DNA contamination
• Compatibility—suitable for use with ExpiCHO and Expi293 expression systems
• Endotoxin-free components— elution buffer, water, and plasmid storage tubes that are free of endotoxin

Simple and fast protocol
Unlike other anion-exchange-based endotoxin-free plasmid purification systems, the PureLink Expi Endotoxin-Free Mega Plasmid Purification Kit utilizes centrifugation- or vacuum-assisted filtration for lysate clarification and subsequent plasmid DNA purification. There is no waiting for gravity-flow columns and the entire purification typically takes 2 hours(including the final precipitation step).

Endotoxin-free plasmid
Purify endotoxin-free (<0.1 EU/µg) plasmid that is ideal for sensitive applications such as transfection of primary cells, genome engineering, and research on gene therapy or plasmid vaccines in vivo.

Compatible with ExpiCHO and Expi293 expression systems
Plasmid purified using the PureLink Expi Endotoxin-Free Mega Plasmid Purification Kit is fully compatible with the Gibco ExpiCHO and Expi293 transient mammalian expression systems.

Superior quality with endotoxin-free components
Rest assured that the kit is of high quality and has additional endotoxin-free elution buffer, water, and plasmid storage tubes for added peace of mind.

RNase A Thermo Scientific™

Thermo Scientific GeneJET RNase A Solution is a component of the GeneJET Plasmid Miniprep Kit (K0502/K0503) and may be purchased separately.

MagMAX™-96 DNA Multi-Sample Kit Invitrogen™

MagMAX™ DNA Multi-Sample 96-Well Kits are designed for rapid purification of high-quality genomic DNA from a variety of sample types including blood and blood cards, tissue, mouse tails, buccal swabs, buffy coats, and cultured cells. These purification kits use MagMAX™ magnetic bead-based nucleic acid isolation technology to produce high yields of purified DNA, free from inhibitors that may affect downstream reactions such as genotyping assays, CE and next-generation sequencing, and high resolution melting analysis. The 96-prep and 5x 96-prep kits are designed for higher throughput and automated format processing.

The demands for DNA yield and purity have become more stringent with the development of new analytical methods and technologies, such as high-throughput genotyping, and next-generation DNA sequencing platforms. MagMAX™ DNA Multi-Sample Kits meet the challenge by delivering highly pure DNA that is free of nucleases, proteins, and other inhibitors of downstream enzymatic reactions.
As shown in Table 1, DNA with high yield and purity can be obtained from a variety of materials using the MagMAX™ DNA Multi-Sample Kits. This is in contrast to filter membrane–based methods that often require different kit configurations for various input materials. For applications requiring maximal yields, for small batch sizes, or for DNA archival purposes MagMAX™ DNA Multi-Sample Kit (tube format, manual processing) may be desirable because larger sample inputs are accommodated. DNA yields suitable for most routine applications may be obtained using the MagMAX™-96 DNA Multi-Sample Kits (96-well plate format, compatible with automated handling) (Table 1).

The MagMAX™ DNA Multi-Sample Kit also delivers high yields of DNA from lipid or glycogen rich samples, such as brain and liver tissues, respectively. Figure 1 shows more than twice as much DNA was purified from tissues with the MagMAX™ DNA Multi-Sample Kit than with the leading competitor’s filter membrane–based method.

Most enzymatic molecular applications are prone to inhibition by analytes carried over from the sample matrix (e.g., heme, heparin, salts, protein) or the purification method itself (phenol, salts, alcohol). Figure 2 illustrates the effect of DNA purity on sensitive molecular applications, such as quantitative PCR (qPCR). DNA was extracted from blood using the MagMAX™-96 DNA Multi-Sample Kit, or a competitor’s filter membrane–based kit and subjected to real-time PCR analysis. The cycle threshold CT values obtained with the DNA purified using the MagMAX™ DNA Multi-Sample Kit were lower than those obtained with the filter membrane kit–purified DNA, indicating the MagMAX™ kit yields higher-purity DNA.

MagMAX™ DNA Multi-Sample Kits provide purified DNA that is suitable for a variety of Applied Biosystems application workflows (Figure 3), including SNP genotyping and DNA sequencing analysis (see Figure 4). To see the full performance description of DNA purified using the MagMAX™ DNA Multi-Sample Kits in TaqMan® SNP genotyping assays and with BigDye® chemistry in DNA sequence analysis, view the MagMAX™ DNA Multi-Sample Kit Product Bulletin.
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