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Lab Automation 2008, January 27–30, Palm Springs, CA
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Poster Sessions: | 1:30 p.m.-3:00 p.m. Jan 28, 2008 |
| 1:30 p.m.-3:00 p.m. Jan 29, 2008 |
Abstract: Amy Yoder, Keith Roby, James Barry--Beckman Coulter Inc. Sharon Soh, Chris Harbert, Francois Degorce--Cisbio International
IP-One HTRF Assay is a homogeneous competitive immunoassay that can be used to monitor Gq-coupled GPCR activity through the inositol phosphate cascade. Inositol Phosphate 1 (IP1) is a downstream metabolite of IP3 that accumulates in cells in the presence of LiCl following Gq-mediated phospholipase C (PLC) activation.
Beckman Coulter, Inc., in collaboration with Cisbio, has adapted the IP-One HTRF assay to an automated liquid handling platform. Detection and analysis was performed using the HTRF cartridge developed for use on the PARADIGM Detection Platform. The target in this study is the Muscarinic M1 receptor, overexpressed in a CHO model system, using carbachol and acetylcholine as reference agonists.
The information in this poster will describe:
Abstract: Li Liu, Amy Yoder and Laura Pajak--Beckman Coulter, Inc.
The reliable and high-quality purification of PCR* products is essential for a successful sequencing process, but manual PCR purification is both labor intensive and time consuming. Therefore, we developed an automated method to provide a complete walk-away system for purification of PCR products. The information included in this poster describes the utilization of the Biomek 3000 Laboratory Automation Workstation for the removal of unincorporated dNTPs, primers, salts and other contaminants from PCR products using Agencourt AMPure reagents. The user interface allows the user to customize an individual run by selecting full or partial 96-well plate and inputting certain reaction parameters.
The information presented here includes the full description of the automated methods and subsequent results. Representative data obtained from the purification of PCR products using this system is shown. Data from capillary sequencing on the CEQTM 8800 Genetic Analysis System from Beckman Coulter demonstrates the suitability of the purified PCR products for stringent assays such as capillary sequencing.
Abstract: Matthew Cu, Mary Blair, Beckman Coulter, Inc.
Isolating templates suitable for sequencing can be challenging particularly with the wide range of vectors used in sequencing today. In the case of manual plasmid preparations, purification is labor-intensive and time-consuming. An automated method was developed for ease-of-use on the Biomek 3000 Laboratory Automation Workstation to isolate templates suitable for capillary sequencing. The automated plasmid-purification protocol employs the Agencourt CosMCPrep kit for high-throughput preparation of high-copy or low-copy plasmids, BAC or YAC DNA from bacterial cultures. The Agencourt CosMCPrep method incorporates a simplified, graphical user interface to allow for sample plate customization. Default settings; such as sample type, number of samples, plate dry time and elution volume; provide a general starting point for the user. The purification process uses alkaline lysis to break open cell membranes followed by Agencourt SPRI® (Solid Phase Reversible Immobilization) technology, which differentially binds plasmid DNA to paramagnetic particles. While the DNA remains affixed to the beads, contaminants can be rinsed away during a washing procedure. After the processing plate is dried, an elution buffer is added to separate the plasmid DNA from the beads. Purified samples can be used in various downstream applications, such as PCR* amplification and fluorescent DNA sequencing.
The information presented here includes the full description of the automated method and subsequent results.
Abstract: Laura Pajak, Mary Blair, Beckman Coulter, Inc.
Low-volume amplification reactions are desirable as a means to deliver high throughput and cost savings. This approach is challenging as it requires accurate dispensing of low-volume sample and reagents into a 384-well reaction plate. Beckman Coulter has automated this process by developing a protocol to dispense master mix using our new BioRAPTR FRD™ Microfluidic Workstation. The BioRAPTR FRD workstation provides a platform that produces highly accurate and precise dispensing while using volumes that range between 100nl to 60μl. This allows for a quick and easy setup of a reaction plate that can contain four to eight different master mixes depending on whether a 4-tip or 8-tip head is being utilized. This protocol was used to generate low-volume standard end-point PCR* reactions. To further test the robustness of the method, STR analysis of human genomic DNA was performed using the GenomeLab™ STR Human Primer kit and CEQ™ 8800 Genetic Analysis System. Genomic DNA was dispensed into a 384-well plate. Master mix was added to the plate using the BioRAPTR FRD workstation. After amplification, the CEQ 8800 Genetic Analysis System was used for separation and fragment analysis.
This poster will describe the process flow for generating low volume amplification reactions for STR analysis and the data obtained from samples processed using these protocols.
Abstract: Amy Yoder, Matthew Cu, Kelly Marshall, and Laura Pajak, Beckman Coulter, Inc.
The Gene Expression Suite of methods was developed to prepare samples for the GenomeLabTM GeXP Genetic Analysis System using the Biomek 3000® Laboratory Automation Workstation. Quantitation and Normalization is the second method in the suite and is the focus of this poster. The Quantitation and Normalization Method is a multipurpose utility designed to quantitate source nucleic acids in a 96-well plate format and normalize the samples to user-defined volumes and concentrations. The method has default parameters for downstream GeXP applications and the user interface allows for customization of many parameters thus permitting the method to be used for applications beyond GeXP sample preparation. The options include nucleic acid type, number of samples, and the choice of quantitation via absorbance at 260 nm or fluorescence using Invitrogen’s Quant-iT* reagents. Standard curve parameters, plate type, and dilutions can also be defined based on the amount and expected concentration of the samples. An integrated calculations template allows for conversion of quantitation data from the DTX 880 Multimode Plate Reader to values to be used by the normalization method. The template records the user defined variables in the Biomek method and allows users to enter their desired normalization parameters after the quantitation calculations are complete. Users can decide how to handle out-of-range samples by either excluding them from further processing or transferring the wells with low concentrations. The method normalizes all wells by volume and concentration with low coefficient of variation.
Abstract: Mary Blair, Kelly Marshall, and Laura Pajak, Beckman Coulter, Inc.
The Gene Expression Suite of Methods for the Biomek® 3000 workstation is designed to quantitate and normalize extracted RNA, prepare a reaction plate for RT-PCR* and PCR, and prepare a sample plate for the GenomeLab™ GeXP Genetic Analysis System. The methods are designed to simplify processing of samples for the GeXP system and can be customized for non gene expression studies. This poster will focus on the last two methods in the suite: Reaction Setup and Plate Preparation.
The Reaction Setup User Interface includes default buttons that allow the user to set up a RT-PCR or PCR plate using the parameters defined by the GeXP kit protocols. The customizable user interface contains options for the samples, master mix, and primers that enable Reaction Setup to double as a general purpose reaction plate setup utility outside of the GeXP system. If a thermocycler is integrated on the workstation deck, the method can move the plate to the thermocycler and execute the amplification protocol providing hands-free processing.
The Plate Preparation method automates the process of creating a buffer plate, dilution plate, and sample plate for processing by the GeXP Genetic Analysis System. Options such as the dilution factor are easily customized via the user interface, allowing the user to optimize each sample set.
* All trademarks are the property of their respective owners. Where applicable,
the PCR process is covered by patents owned by Roche Molecular Systems, Inc.,
and F. Hoffmann-LaRoche, Ltd.
† Application under development
For Research Use Only; not for use in diagnostic procedures.
For comments or questions about T3 Update, please contact the T3 Editor.
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