Automated Purification of Plasmid DNA Using Paramagnetic Particles

We describe a reagent system and robotic method for purifying plasmid DNA for restriction digestion, PCR, and fluorescent sequencing applications. The method uses two types of Wizard® MagneSilTM paramagnetic particles. Following lysis and neutralization procedures, the first particle type binds and removes cell debris; the second type is then used to bind plasmid DNA from the cleared lysate. The particles are then washed to eliminate unwanted contaminants. Purified plasmid DNA is then eluted from the particles with nuclease free water. When using a cell mass of approximately 4 O.D.₆₀₀, the yield is 10–12μg of DNA when using high copy number plasmid. When used in BigDye® terminator sequencing, these DNA templates typically yield read lengths greater than 700 bases and Phred 20 scores of 600 to 750 bases. This purification method has been adapted for use on several robotic platforms in a 96-well format.     

A Fully Automated High-throughput Nucleic Acid Purification System Using Silica Coated Magnetic Bead Technology

The design of a fully automated high-throughput system for the purification of sequencing templates is described. Hardware, software, and chemistries have been optimized to suit the needs of high-throughput laboratories involved in genome sequencing projects. Using this system, up to 5760 samples (60 x 96-well plates) can be purified in less than 16 hours during a single unattended run. The system can also be configured to perform sequencing reaction setup for all 60 plates following template purification, extending total run times to < 24 hours. Final sequencing reactions are prepared in 384- well microplates.     

MagneSil™ Paramagnetic Particles: Magnetics for DNA Purification

MagneSil™ Paramagnetic Particles are silica-paramagnetic particles with an affinity for nucleic acids under defined conditions. Particle structure and solution composition can be altered to selectively adsorb nucleic acids based upon type and size. These properties have been used to develop purification methods based on a three-step bind, wash, and elute process. The MagneSil™ technology is readily adaptable to robotic platforms, allowing complete automation of the purification process in either 96- or 384- well plate formats. This article introduces the basic physical and performance characteristics of the MagneSil™ Paramagnetic Particles.     

Developing an Automated Enzyme Immunoassay for High-Throughput Screening of Bovine Serum Antibodies to Neospora Caninum

An enzyme-linked immunosorbent assay (ELISA) for Neospora caninum antibodies was automated with a robotic workstation, the Beckman Coulter Biomek 2000, to screen 200 bovine sera. Comparing these results with manually run ELISA data, a 95.92% agreement (K = 0.9592) between the two assays was obtained. The automated assay was specific and sensitive with excellent positive and negative predictive values. The results were repeatable and reproducible. The automation flexibility was high and the operation complexity was minimal. High-throughput screening (HTS) for bovine antibodies to Neospora caninum was achieved. The assay was developed according to the internationally recognized ISO17025 standard requirements.     

Automated High-Throughput Purification of PCR Products Using WizardMagneSil™ Paramagnetic Particles

Here we describe a reagent system and robotic methods for the purification of PCR^(a) fragments from other contaminating amplification reaction components. The methods use the MagneSil™ paramagnetic particle chemistry^(b) to isolate double stranded DNA fragments from 150bp to 23kbp. Purified fragments are eluted in water ready for downstream applications such as cloning, fluorescent DNA sequencing and microarray printing. This method has been adapted to a number of liquid handling robotic platforms, including the Biomek^® FX and Biomek^® 2000 Laboratory Automation Workstations, in both 96 and 384-well formats.     

Automated Lab-on-a-Chip Analysis of DNA Fragments

The design of a fully automated system for the analysis of DNA fragments in 96-well plates is described. Microfluidic technology is used to integrate sample loading, electrophoretic analysis, and fragment detection onto a miniature lab-on-a-chip device. The microfluidic chip operates in an instrument platform that automates sample access, data collection, and data reporting. Each microfluidic chip provides sizing and concentration values for more than 1000 DNA samples.     

Automated Purification of DNA from Large Samples: A Study of Effectiveness and Labor Efficiency

Investigations into the underlying genetic contributions to human disease are transitioning from small family-based traditional linkage analyses to large population-based studies designed to identify genetic factors in more complex and common diseases that have the greatest impact on human health. These types of studies have driven the need for larger numbers of samples for analysis and more efficient and effective methods for DNA purification, especially for large samples that provide sufficient quantities of DNA for extensive analysis. The AUTOPURE LS™ Nucleic Acid Purification Instrument, by Gentra Systems, Inc., a platform capable of high-throughput sample purification from large samples, was developed to meet the demands of these large studies. This article presents data demonstrating the equivalency of DNA purified using the AUTOPURE LS automated instrument and the manual method based on the same purification process. In addition, we present data demonstrating the in-lab time savings realized by automating the purification process.     

The First Automated High Content Screening System

With biological discovery and technology advancing in parallel, AstraZeneca (AZ) announced that it would soon take delivery of what is thought to be the first fully automated high-throughput high-content screening system. This custom designed assay platform from RTS Life Science International (RTS) automates the IN Cell Analyzer 3000 sub cellular analysis system from Amersham Biosciences. RTS has integrated its advanced scheduling system with the imaging tool to enable AZ to evaluate the effect of drug compounds on cellular processes.     

Automated Nucleic Acid Purification for Large Samples

Advancements in the fields of genomic screening, molecular pathology and clinical research have resulted in a major increase in the demand for high quality DNA and RNA. This escalating demand has resulted in a sample preparation bottleneck and an emphasis on the development of new technologies to automate the purification process. Gentra has developed the AUTOPURE LS™ nucleic acid purification instrument, a platform capable of high-throughput sample purification from large samples, such as 10 mL whole blood. This article presents data showing the equivalency of DNA purified using manual and automated processing.     

Controlling a Twister II with VBA and the Zymark ZyRobot ICP

A Visual Basic for Applications program was developed for direct control of a Twister II robot using the Zymark ZyRobot_ICP. Direct control enables automated check-out of the robot, and automated measurement of the plate stacks prior to a robot run. Normal operation of the robot program is performed with just two buttons.     

Automated RNA Isolation Using the Ambion RNAqueous™-96 Kit with the Packard MutiPROBEII HT Liquid Handling System

Automated isolation of total RNA from cultured cells was accomplished by adapting Ambion's solid-phase total RNA isolation kit, RNAqueous-96, for use with the MultiPROBE II liquid handling system from Packard Instruments.Cultured cells (HeLa S3 adherent cells and K562 cells in suspension) were lysed, DNase treated, washed, and then eluted from the 96-well filter plate.Total RNA recovered was analyzed for yield, intactness, purity, and ability to serve as a substrate for real-time RT-PCR.     

Transferring Industrial Automation Technology to the Laboratory

Over the past few years, there has been much discussion about transferring industrial technology to laboratories. While it is easy to look at the superficial similarities, it is more important to examine the different requirements of different industries. In this way, it is possible to identify the technologies and techniques that can be successfully transferred to the laboratory to improve performance.This paper takes three very different industries - the bakery, High Throughput Screening (HTS) and mobile phone assembly and examines their different requirements. These industries have been selected from among the many sectors where the RTS Group operates - thus allowing real data from a number of situations to be used.One of the most important areas in automation design is the relationship between flexibility and throughput. This paper focuses on this relationship and its influence over machine configuration when comparing the requirements of the different industries.     

Detection of Cellular Parameters Using a Microfluidic Chip-Based System

Lab-on-a-chip technology achieves a reduction of sample and reagent volume and automates complex laboratory processes. Here, we present the implementation of cell assays on a microfluidic platform using disposable microfluidic chips. The applications are based on the controlled movement of cells by pressure-driven flow inside networks of microfluidic channels. Cells are hydrodynamically focused and pass the fluorescence detector in single file. Initial applications are the determination of protein expression and apoptosis parameters. The microfluidic system allows unattended measurement of six samples per chip. Results obtained with the microfluidic chips showed good correlation with data obtained using a standard flow cytometer.     

Automated Lead Discovery and Development in HTS Datasets

The analysis of biological screening results has traditionally been a labor intensive process. Scientists familiar with the biological data under investigation would visually inspect the results, evaluate the quality and promise of active data points and identify leads. The introduction and widespread use of high throughput screening systems has increased the size of biological datasets immensely thus pushing the traditional analysis method to its limits. In this presentation we describe a new automated approach that emulates the decision making process of human experts. This approach combines knowledge-based techniques with human expertise to enable rapid identification, characterization and prioritization of lead candidates.     

An Automated System for Processing Sample Vials Stored at Ultra-Low Temperatures

The BIOPHILE Individual Vial Retriever (IVR) system has been developed to provide automated access to vials stored at ultra-low temperatures. The IVR performs storage, retrieval, sorting, cataloging, volume estimation (weighing), barcode reading, and re-racking. All racking operations are performed in an environment designed to keep samples frozen at their optimal temperature. Operating temperatures are −80°C, −40°C, −20°C, and room temperature. Laboratory Information Management System (LIMS) integration, automation integration, chain of custody tracking, and FDA 21CFR Part 11 compliance are supported. This article introduces the IVR and provides information on its characteristics.     

A Fully Automated Process Using a Magnetic Particle Based Kit for Removal of Dye Terminators from Sequencing Reactions

Prolinx,® Inc. of Bothell, WA has developed the RapXtract™ 384 Dye Terminator Removal Kit for full automation of DNA sequencing reaction purification. The RapXtract product line is based upon proprietary superparamagnetic particle technology that eliminates the need for centrifugation, vacuum filtration, or modified primers to achieve purification of sequencing reactions. The kit described here is pre-dispensed in a 384-well microtiter plate and run on the TECAN GENESIS Workstation 150 (Tecan U.S. Inc., Research Triangle Park, NC). This system enables rapid purification of up to 384 sequencing reactions in a single run.As the completion of the Human Genome Project nears, it is imperative for biotechnology and pharmaceutical companies to increase throughput of DNA sequencing in order to be competitive in the drug discovery and validation process. The “race to market” requires a shift from standard DNA sequencing processes-including DNA sequencing reaction purification-towards complete walk-away automation for all steps.Existing sequencing reaction purification methods (Table 1) require considerable resources including: plastic and other laboratory consumables; specialized equipment, such as high-speed centrifuges or vacuum filtration apparatus; and labor-intensive protocols requiring large amounts of technician time. As a result, walk-away automation of standard purification methods is difficult and expensive.     

Development of an Automated High-Throughput Analytical Characterization System for Support of a Central Sample Collection Resource

A high-throughput analytical characterization system was developed for quality control support of a central sample collection resource. This system utilizes liquid chromatography mass spectrometry with in-house developed data automation applications. Continuous operation of analytical instrumentation is accomplished by fully automating sample submission and report processing functions. Comprehensive analytical information characteristic of quality, chemical, and physical properties (e.g. relative purity, detection sensitivity, LogD) are automatically transferred to an on-line database. The application of this database for detailed quality assessment of a small sample library (ca. 24,000 compounds) is demonstrated.     

Incorporating Automation in Undergraduate Laboratories

Automated techniques and philosophies, which have become increasingly important in modern laboratories, are not traditionally covered in undergraduate chemistry curricula. To this end, we have been incorporating automated sample preparation methods using standard robotic workstations in our undergraduate analytical chemistry laboratory. Using a Benchmate™ II Workstation, an automated method has been developed and implemented for the solid phase extraction of capsaicins from commercial hot pepper sauces prior to liquid chromatographic analysis. This paper reports on pedagogical aspects of incorporating automation into the undergraduate curriculum as well as results obtained for manual and automated extractions conducted by students.     

Development of a μ-concentrator Using Dielectrophoretic Forces

We present the simulation, development and experimental validation of a μ-concentrator based on dielectrophoresis, DEP.In a first step dielectrophoretic force fields of various electrodes are computed and compared. The simulation results for various electrode dimensions may serve as a general design rule for DEP devices. Favorable electrode designs were realized in gold on glass substrates. The performance of the DEP chips is validated by concentration of E.-Coli bacteria, a separation efficiency of 99.93% was achieved. Furthermore, we outline how the combination of forced convection and DEP allows for bacteria separation at increased flow rates.