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.     

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.     

High Throughput Methods for Gene Identification, Cloning and Functional Genomics Using the GeneTAC™ G Robotics Workstation

Using a single robotic platform, the GeneTAC™ G³, we have automated most of the processes involved in the cloning and characterisation of novel disease causing genes by addressing the following; firstly, identifying the BACs of interest and making shotgun libraries. Secondly, automating the set up of sequencing reactions using methodology that eliminates the need for DNA preparation of 384 clones. Thirdly, generating sublibraries using selective re-arraying of library clones to enable the determination of the entire genomic sequence of the gene. Fourthly, determining gene function by combination of differential screening and mini Northerns using microarrays printed using the GeneTAC™ G³ system and hybridised using the GeneTAC™ HybStation (Genomics Solutions, Ann Arbor, USA).     

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.     

Creating change and driving innovation in highly automated and lean organizations: The Temporal Think Tank™ (T3™)

This paper introduces the concept of the temporal think tank™ (T3™), a temporary in-house research center, based on case studies with a highly automated global manufacturer and in various small-sized manufacture (SME's). The T3™ brings individuals from across the organization together in a team setting in which they are charged to look at the future, develop their decision-making skills, and use entrepreneurial thinking to incubate new processes and/or products. After an appropriate time in the temporal think tank™, the individuals are asked to return to their regular positions to implement the best practices and best ideas and doing so spur the organization forward. At intervals, the temporal think tanks™ can be reconvened, or reconstituted with new participants. This approach prepares technology champions for the SME, who can return to the organization with a fresh outlook and renewed energy to keep the enterprise at the peak of efficiency and effectiveness.     

“Electric” Zip Tips™, Preliminary Results

Induction based fluidics (IBF), a new, simple patented approach for transporting liquids in the micro and the macro world, is discussed. Electric fields are shown to energize liquid/s in a container/s to execute an array of purposes. IBF is shown uniquely to energize N liquids in simple off the shelf devices, inductively. We discuss calibration and other issues, as we demonstrate how simple devices can dispense nanoliters and microliters with high precision and accuracy. Furthermore, we show preliminary single and eight channel data for the Zip Tip™ made by Millipore where the device transports liquids “electrically.” We briefly consider how such new devices, “electric” Zip Tips™, might automate desalting and the placement of digests for MALDI TOF analysis.     

Automated Purification of Transfection-Grade Plasmid DNA Using Wizard MagneSil Tfx System

We describe a reagent system and robotic protocol for the isolation of highly purified plasmid DNA from cultured cells. The method is based on the Wizard® MagneSil™ Plasmid Purification System, which purifies sequencing-grade plasmid DNA. Two modifications to the sequencing-grade system were made to create the Wizard MagneSil Tfx™ System. This system provides lower endotoxin and other contaminant levels, giving higher quality plasmid for transfection applications. The Wizard MagneSil Tfx™ System uses MagneSil™ Paramagnetic Particles (PMPs) to clear lysate and bind plasmid, eliminating the need for filtration devices. The endotoxin removal step uses MagneSil™ (PMPs) and a guanidine/isopropanol wash to remove RNA and protein. One 96-well plate may be processed in 45 minutes on the Beckman Biomek® FX robotic workstation. We provide data showing DNA yield, contaminant levels, and transfection efficiency for 5 commonly used cell lines. Comparisons with other systems are also shown.     

A 384-Well, Fully-Automatable, Superparamagnetic-Particle Based Dye Terminator Removal Kit

We have developed a method for the automated purification of DNA sequencing reactions using the RapXtract™ 384 Dye Terminator Removal Kit and the Quadra 3™ Workstation. The process enables purification of 384 reactions in five minutes, significantly impacting the through-put potential of sequencing laboratories. The RapXtract technology utilizes superparamagnetic particles, (i.e., particles that are not themselves magnetic but that respond to a magnetic field) and eliminates the need for centrifugation, vacuum filtration, or modified primers. The Quadra 3 Workstation is a 384-channel liquid handling system, fitted with a retractable magnetic nest designed to incorporate a 384 magnetic separator. The combined technologies result in reduced variability associated with manual methods for sequencing reaction purification.     

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.     

The Synergy™ HT - A Unique Multi-Detection Microplate Reader for HTS and Drug Discovery

Pharmaceutical and biotechnology research requires instrumentation to be both functional and versatile. In the HTS and Drug Discovery environments, microplate based assays are developed to make determinations on large numbers of samples. Regardless of the assay protocol, the end result is the measurement by some sort of detection device. Towards that end, Bio-Tek has developed the Synergy™ HT Multi-Detection microplate reader. Microplate Reader. Synergy HT utilizes two independent sets of optics to provide uncompromised performance. For absorbance measurements, there is a xenon-flash lamp with a monochromator for wavelength selection and photodiode detection. This allows the selection of any wavelength for endpoint or kinetic measures from 200 nm to 999 nm in 1 nm increments, as well as spectral scans. Traditional visible wavelength fluorescence measurements are made using a tungsten-halogen lamp with interference filters (excitation and emission) for wavelength selection and photomultiplier (PMT) detection. Glow luminescence measurements are also easily accomplished in Synergy HT. If time-resolved or UV excitation fluorescence measurements are required, Synergy HT automatically integrates the xenon-flashmonochromator excitation with the interference emission filter and PMT detection. Typical applications include antibody-antigen binding, receptor-liquid binding, ELISA, nucleic acid quantitation using fluorescent dyes or direct UV analysis. Synergy HT is capable of reading any plate format up to 384-well plates and provides temperature control up to 50° C and shaking as standard features. It is compact (16″ W × 15″ D × 10″ H) and robotics-compatible through the OLE functionality of the KC4™ data reduction software that is bundled with the instrument. An upgrade to CFR 21 Part 11 compliant software is also available.     

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.     

Automated High Throughput Screening of Fluorescent Imaging Plate Reader (FLIPR) Assays Using Assay Platform™ Integrated with Activity Base for ‘on the fly’ Compound Reconfirmation

Advances in the field of automation have meant hitherto complex manual cell-based assays can now be automated. These improvements have brought significant enhancements in throughput, data fidelity and consistency, and allowed a reallocation of constrained resources.Building upon these improvements, we have linked our automated cell-based screening system, Assay Platform™, to Activity Base (IDBS), a software package designed to automate the analysis of HTS data. Customisation of this package has resulted in software that can identify ‘active’ compounds and re-pick them ‘on the fly’ from the original compound plates for triplicate re-testing without operator intervention.Based on an operator initially defining ‘normal’ parameters for assay activity in Activity Base, combined with an automated quality control software module that checks data fidelity, wells containing ‘active’ compounds can be re-picked and re-tested at the end of an automated screening run. Automating cell-based assays has significantly improved productivity, and, with the synergism of Activity Base, has given us greater power to complete each screening run and report ‘active’ compounds to Chemistry more rapidly. This article presents our approach to the automation of cell-based Fluorescent Imaging Plate Reader (FLIPR) screening together with automated active re-test confirmation using Activity Base.     

An assembler and disassembler framework for Java™ programmers

The Java™ programming language is primarily used for platform-independent programming. Yet it also offers many productivity, maintainability and performance benefits for platform-specific functions, such as the generation of machine code.

We have created reliable assemblers for SPARC™ , AMD64, IA32 and PowerPC which support all user mode and privileged instructions and with 64 bit mode support for all but the latter. These assemblers are generated as Java source code by our extensible assembler framework, which itself is written in the Java language. The assembler generator also produces javadoc comments that precisely specify the legal values for each operand.

Our design is based on the Klein Assembler System written in Self. Assemblers are generated from a specification, as are table-driven disassemblers and unit tests. The specifications that drive the generators are expressed as Java language objects. Thus no extra parsers are needed and developers do not need to learn any new syntax to extend the framework for additional ISAs.

Every generated assembler is tested against a preexisting assembler by comparing the output of both. Each instruction’s test cases are derived from the cross product of its potential operand values. The majority of tests are positive (i.e., result in a legal instruction encoding). The framework also generates negative tests, which are expected to cause an error detection by an assembler. As with the Klein Assembler System, we have found bugs in the external assemblers as well as in ISA reference manuals.

Our framework generates tens of millions of tests. For symbolic operands, our tests include all applicable predefined constants. For integral operands, the important boundary values, such as the respective minimum, maximum, 0, 1 and −1, are tested. Full testing can take hours to run but gives us a high degree of confidence regarding correctness.     

Corning® 384 Well Low Volume Microplate Performance in Miniaturized Assays

In response to the ever-increasing demand for microplates that facilitate assay miniaturization without compromising assay integrity, Corning's new 384 well Low Volume (384 LV) microplates were evaluated using a homogenous, fluorescence polarization protease assay. Fluorescently labeled casein substrate was incubated over time with dilutions of protease at working volumes that range from 1 to 20 μL and reagent concentrations that range from 0.125 to 50 pg/μL. Four types of plates were evaluated:

• Corning 384 well LV black medium bind

• Corning 384 well LV black non-binding surface (NBS™)

• Corning 1536 well black medium bind

• Competitor 384 well small volume

Protease activity was detected as a decrease in polarized fluorescence resulting from substrate degradation over time. Following assay miniaturization to 20 μL working volumes and protease concentration reduction from 25 to 0.25 pg/μL, Corning 384 well LV medium bind and NBS coated assay plates maintained superior signal to noise ratios compared to competitor's 384 well small volume assay plates. At even lower 10 μL working volumes, Corning 384 well LV medium bind assay plates maintained signal to noise ratios comparable to the competitor's assay plates. However as the reagent concentrations were further reduced to 2.5 pg/μL, Corning 384 well LV medium bind and NBS coated assay plates had significantly greater signal to noise ratios compared to the competitor's assay plates. As working volumes were reduced even further to 5 μL and 1 μL, Corning 384 well LV medium bind and NBS coated assay plates had significantly greater signal to noise ratios than the competitor's 384 well small volume assay plates. Furthermore, at 1 μL working volumes, Corning 384 well LV NBS coated assay plates had significantly greater signal to noise ratios than the competitor's assay plates and standard 1536 well assay plates.These results suggest that Corning 384 well LV medium bind and NBS coated assay plates facilitate assay miniaturization while maintaining assay integrity. Furthermore, as working volumes were significantly reduced to those typically found in 1536 deep well and standard 1536 well assay plates (5 and 1 μL, respectively), Corning's 384 well LV NBS coated assay plates provided superior assay performance.     

Dispensing Precision for the SciClone™ Automated Liquid Handling Workstation for 96-Channel Pipetting

One of the first steps in drug discovery involves identification of novel compounds that interfere with therapeutically relevant biological processes.

Identification of ‘lead’ compounds in all therapeutic areas included in a drug discovery program requires labor-intensive evaluation of numerous samples in a battery of therapy targeted biological assays. To accelerate the identification of ‘lead’ compounds, Janssen Research Foundation (JRF) has developed in the past an automated high throughput screening (HTS) based on the unattended operation of a custom Zymark tracked robot system. Automation of enzymatic and cellular assays was realized with this system adapted to the handling of microtiter plates. The microtiter plate technology is the basis of our screening. All compounds within our chemical library are stored and distributed in micronic tube racks or microtiter plates for screening. An efficient in-house developed mainframe based laboratory information management system supported all screening activities. Our experience at JRF has shown that the preparation of test compounds and serial dilutions has been a rate-limiting step in the overall screening process. In order to increase compound throughput, it was necessary both to optimize the robotized assays and to automate the compound supply processes. In HTS applications, one of the primary requirements is highly accurate and precise pipetting of microliter volumes of samples into microplates. The SciClone™ is an automated liquid handling workstation capable of both 96- and 384-channel high precision pipetting. For high throughput applications, the SciClone™ instrumentation is able to pipette a variety of liquid solutions with a high degree of accuracy and precision between microplates (inter-plate variability) and tip-to-tip (intra-plate variability) within a single plate. The focus of this presentation is to review the liquid handling performance of the SciCloneTM system as a multipurpose instrument for pipetting aqueous or organic solutions, and virus suspensions into 96- and 384-well microplates. The capabilities of the system and the resulting benefits for our screening activities will be described.     

Simlab 1 - A Laboratory Workflow Recorder for Process Assessment

Process management and workflow optimisation have become key issues of good laboratory management and now seem to be predominant over traditional challenges such as the quality of analytical testing. The first author of this paper has conducted several workflow optimisation studies for clinical laboratories in Europe (Universities of Zurich and Amsterdam, Manchester Royal Infirmary and others) and in the US (University of Virginia), using a laboratory-specific simulation software called Simlab™ for realistic computer modeling of laboratory scenarios. Since data input and calibration of the models against reality have always been a challenge in these studies, we looked into possibilities of using automated data extraction to extract as much information as possible from the laboratory information system. In this paper we describe our first experience with a Microsoft Excel™-based software program called Simlab 1, which uses the ASCII file transfer protocol to extract data from the LIS.     

The LabMatrix system

The LabMatrix^™ is a prototyping system designed to give the user a practical and versatile platform for testing microfluidic applications in the fields of health care and life sciences. The LabMatrix^™ system consists of a microfluidic breadboard and cover that align and secure a series of specially designed LabMatrix^™ microfluidic chips. Chips are easily arranged and rearranged into a user-defined fluidic network. The LabMatrix^™ system is designed with maximum flexibility in mind, providing the user with a means to prototype a wide range of microfluidic applications in a short period.     

Sequencing and Scheduling in Robotic Cells: Recent Developments

A great deal of work has been done to analyze the problem of robot move sequencing and part scheduling in robotic flowshop cells. We examine the recent developments in this literature. A robotic flowshop cell consists of a number of processing stages served by one or more robots. Each stage has one or more machines that perform that stage’s processing. Types of robotic cells are differentiated from one another by certain characteristics, including robot type, robot travel-time, number of robots, types of parts processed, and use of parallel machines within stages. We focus on cyclic production of parts. A cycle is specified by a repeatable sequence of robot moves designed to transfer a set of parts between the machines for their processing.We start by providing a classification scheme for robotic cell scheduling problems that is based on three characteristics: machine environment, processing restrictions, and objective function, and discuss the influence of these characteristics on the methods of analysis employed. In addition to reporting recent results on classical robotic cell scheduling problems, we include results on robotic cells with advanced features such as dual gripper robots, parallel machines, and multiple robots. Next, we examine implementation issues that have been addressed in the practice-oriented literature and detail the optimal policies to use under various combinations of conditions. We conclude by describing some important open problems in the field.