Run PCR experiments in triplicate (various module/experiment combinations are utilized to make 96-well plates).Design PCR experiment and capture relevant details in tracking database.The workflow is comprised of the following steps: In practice, the PCR Test Panel involves a typical PCR workflow from experimental planning to data analysis, with crucial steps of database integration. Importantly, this system is easily adapted and can be scaled to assess new components and conditions as they arise.
Some of the variations in template and primer components that are assessed by the PCR Test Panel are described in Table 1. Table 1: Examples of Test Panel Variablesīy varying large numbers of conditions and quantitating both specificity and yield of a PCR product, the contribution of each reaction component can be systematically evaluated. Cycling conditions, thermocycler ID and other relevant details are also tracked.
A supporting, internal database has been designed to link the results from the microfluidic analysis (e.g., yield and purity of each expected product) to the detailed contents of each well (e.g., identity and concentration of polymerase, template, buffer, additives, etc.). Use of the PCR Test Panel is simplified by a microfluidic, agarose gel-based mimic (LabChip® GX platform, Caliper Life Sciences, Hopkinton, MA) that allows rapid end-point quantitation of amplification reactions in a 96-well plate format. Facilitate the development of enhanced tools (e.g., polymerases, buffers, etc.) that will broaden the definition of "routine PCR" to encompass situations that are currently challenging.Provide information to enable researchers to modify a limited set of variables depending on the desired outcome (or inherent limitation) of their particular set of amplifications.Understand the contributions of each variable to the desired signal-to-noise outcome.Systematically manipulate the numerous variables present in an amplification reaction.As part of these efforts, we have created a quantitative, microfluidic-based, PCR Test Panel. With the long-standing goal of enabling the research of our customers and our own scientists, NEB continues to devote resources both to basic DNA polymerase research and to the development of new products for DNA amplification. However, practical issues, such as very high (or low) template GC content, the presence of inhibitors, limitations of primers, source materials or time, can limit the ability either to follow optimized PCR guidelines or to systematically evaluate a sufficient number of variables to ensure success. Ideally, various components can be altered to achieve success and even to favor a desired outcome (e.g., specificity over yield, sensitivity over specificity, etc.). It is only after unsuccessful amplification attempts that these variables become evident. The range of variables that impact DNA amplification reactions is masked by the relatively high success rate of PCR experiments.
By understanding the interplay of these variables, PCRbased tools and techniques are bolstered, and difficult amplifications become routine. Success or failure in PCR is influenced by a myriad of factors including primer design, cycling conditions, and the quality and concentration of reaction substrates and solutions. As such, it has significantly impacted research and development in fields such as biochemistry, medicine, bioengineering and beyond. PCR (1) is arguably the most common technique in molecular biology.
0 kommentar(er)
