E. coli is often chosen as a host for recombinant protein expression because it’s easily manipulated and produces high yields of recombinant protein. Obtaining sufficient recombinant protein is often one early step in a long research project, so progressing quickly from cloned or synthesized cDNA to soluble, active protein is essential. Whether your lab is expressing a single target or multiple targets in a high-throughput screen, common challenges such as toxicity, low solubility or expression levels, and inefficient fusion tag cleavage can slow or stop progress. In this webinar we will discuss scalable solutions to speed and simplify protein expression at every step, including cloning, expression, and testing of protein yield and solubility. We will discuss novel fusion tags that enhance solubility, and tips and tricks for efficient fusion tag cleavage. We will also introduce methods to express difficult targets, and present case studies illustrating toxic or insoluble protein production from cloning through functional characterization.
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|Presented by |
|Moderated by |
Karen Kleman, Ph.D.,
Aired Wednesday, 8 March 2017
Fred Hyde, Ph.D. Staff Technical Applications Scientist, Illumina, Inc.
Have you ever generated libraries of mutants or gene knockouts, but struggled to get complete representation in your library? Do you need techniques to help easily identify essential genes or regulatory elements? Perhaps you have struggled with de novo sequencing of large genomes, or rescuing plasmid DNA from environmental samples in metagenomics screens? In this webinar, we will talk about some of the challenges inherent in these applications, and how transposons, or “jumping genes”, can help. We will introduce EZ-Tn5 transposon mutagenesis* as a well-known, easy-to-use system that employs an engineered, hyperactive Tn5 Transposase enzyme that is 100 times more active than the native Tn5 transposase. We will discuss how transposons are used by many researchers for metagenomics, bacterial strain development and gene knockout mutagenesis in vitro and in vivo. We will also present techniques for introducing mutations in bacterial genomes using simple electroporation, “rescuing” non-coliform plasmid DNA from environmental organisms, and creating custom transposons using PCR for novel applications. In addition, we will address customer questions and provide troubleshooting tips.
Key Learning Objectives
* These Epicentre-branded kits are now exclusively available through Lucigen.
Aired Wednesday, 25 January 2017
Robert Brazas, Ph.D. Senior Product Manager, Lucigen
Have you ever wanted to analyze your favorite genomic DNA (gDNA) sample, but didn’t have enough starting material? Perhaps you wanted to perform whole genome sequencing on a single mammalian cell (e.g. a single cancer cell), but couldn’t effectively make your next-gen sequencing fragment library because you didn’t have enough DNA? Or maybe you had enough material from your metagenomic sample to perform one PCR analysis, but you used up the entire sample in that one experiment and couldn’t archive any material for future analyses? If you’ve experienced any of these or other issues due to limiting gDNA samples, then this webinar will help you identify potential solutions. We’ll introduce whole genome amplification using multiple displacement amplification (MDA), a technique which enables high-fidelity production of micrograms of gDNA from femtograms of gDNA. Specifically, this webinar will (i) provide an overview of how MDA works, (ii) introduce the various forms of MDA (random priming vs. enzymatic priming), and (iii) compare the performance of various MDA kits. The data presented will include next-gen sequencing results using whole genome amplified DNA as starting material. Together, this webinar will help you understand the key differences between the various MDA kits/approaches and enable you to choose the most appropriate kit for your experiments with precious, limiting gDNA samples.
Key Learning Objectives
Aired Wednesday, 9 November 2016
Presented by Dipankar Manna, Ph.D., Principal Scientist, R&D, Lucigen
Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification method that is rapid, sensitive, accurate, and cost-effective. Due to the isothermal amplification characteristics, LAMP is amenable to assaying diagnostic samples at the point-of-care or environmental samples at the point-of-collection. Over the years scientists have used LAMP technology to develop assays for a variety of infectious agents including bacteria, viruses, fungi and parasitic organisms. They also developed assays for the diagnosis of cancer, identification of invasive species, detection of food adulteration and identification of drug resistance, among others. However, assay development for new targets remains challenging in part due to the complex design requirement of six LAMP primers and the need to optimize buffer conditions. Suboptimal assay design results in slow amplification kinetics, low sensitivity, and poor specificity. This webinar will outline the basic design of LAMP assays and how it is different from PCR. We will discuss key parameters that affect assay performance and approaches to assay optimization. We will also detail common methods to monitor amplification and how to troubleshoot assay failures. Lastly, we will introduce a novel thermostable enzyme suitable for robust and sensitive DNA as well as RNA LAMP.
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Originally aired Wednesday | September 14, 2016
Presented by Beth Frey, Product Manager, Lucigen and moderated by Karen Kleman, Ph.D., Technical Support & Applications Scientist, Lucigen
E. coli is a cell-factory for producing large quantities of recombinant protein for basic research studies, industrial bioprocesses, drug discovery, and therapeutics. Although a variety of heterologous proteins from viral to human origin have been successfully overproduced in bacterial systems, each new gene sequence and encoded protein can present well-known expression challenges including low expression levels, protein misfolding, and E. coli cellular toxicity. In this webinar you’ll learn the cellular mechanisms that lead to these impediments and expression strategies for overcoming them. We will introduce enhanced E. coli expression systems with optimized vector properties and unique promoters for tunable induction control and high-level expression. Additionally, we will discuss novel fusion tags that enhance protein expression and solubility, and a 5-second, enzyme-free cloning system that facilitates parallel processing and high-throughput expression screening. You’ll also pick up helpful bench tips for generating soluble and active protein ready for different downstream applications.
Key Learning Objectives
Explore the root causes of common protein expression failures in E.coli.
Learn how stringent growth conditions combined with tunable promoters and novel fusion tags improve protein expression and solubility.
Discover ways to streamline your protein expression screening workflow with a flexible, enzyme-free cloning system.
Competent Cells 101: Maximizing Your Success in Cloning, Protein Expression, and Library Generation
Originally aired Wednesday, 13 July 2016
Presented by Eric Steinmetz, Ph.D., Principal Scientist, R&D, Lucigen
Competent E. coli cells are ubiquitous in biological research. They’re used for routine cloning and propagation of plasmid DNA as well as for the construction of complex libraries with very high diversity used in drug discovery processes and other screening applications. Although competent cells are often taken for granted, selecting the best strain and most appropriate format for your application is critical for success. In this webinar, you’ll learn how to choose the best strain and cell format and pick up technical tips for maximizing transformation efficiency and experimental success. You’ll also discover specialized strains for lentiviral CRISPR libraries, phage display, production of DNA and protein with low endotoxin, and cloning of difficult or “unclonable” DNA.
Clone the Unclonable - Vectors and Cells to Capture and Express Problematic DNAs
Originally aired Wednesday, 11 May 2016
Presented by Ron Godiska, Ph.D., Senior Scientist, R&D, Lucigen
Almost every molecular biologist has encountered DNA targets that are unstable or extremely difficult to clone in E. coli. Common examples include repetitive sequences, AT-rich regions, or viral genes that encode toxic proteins.
In this webinar we will highlight cloning vectors and E. coli strains specifically developed to facilitate cloning of targets that cannot be captured in common vectors or cells. For example, we will demonstrate how the novel pJAZZ linear vectors and the CloneSmart circular vectors enabled cloning and expression of long repeats, centromeric DNA, AT-rich libraries, and other difficult targets. We will also introduce simplified techniques for cloning fragments with no ligation or other enzymatic treatments. Finally, we will highlight E. coli strains which enable expression of toxic as well as difficult membrane proteins.
This webinar will cover the following topics regarding cloning difficult DNA targets:
Next Gen Sequencing: Library Prep Challenges and Solutions
Originally aired Wednesday, 9 March 2016
Presented by Robert Brazas, Ph.D., Next Gen Sequencing Product Manager, Lucigen
You’re most likely interested in next gen sequencing (NGS) for a reason; you want big data! To get that big data, it’s important to optimize each step in the NGS workflow, otherwise data quality and quantity will suffer. This webinar will focus on a key step in next gen sequencing of DNA, fragment library construction. Problems encountered at this step can dramatically impact your results, and this webinar will outline the potential issues encountered during library prep and provide solutions. Specifically, we will introduce the new NxSeq® AmpFREE Low DNA Library Kit which solves many of the common library prep challenges such as inefficient library construction, limiting amounts of starting material and PCR-bias. We will also discuss the construction and use of mate pair libraries for generating the long-range linkage information crucial for scaffold improvement during genome assembly as well as transgene/virus genome insertion mapping and chromosomal structural variation identification.
Originally aired Tuesday, 9 February 2016
Presented by Dr. Armin Schneider, SVP Research, and Dr. Angel Picher, Associate Director, Product Development, Sygnis. Lucigen is an official United States distributor of Sygnis products.
Single cell whole genome amplification using MDA (multiple displacement amplification) relies on priming by random hexamers, which can result in amplification bias. Sygnis’s revolutionary TruePrime™ technology uses a combination of Phi29 DNA polymerase and the recently discovered primase/polymerase TthPrimPol to uniformly amplify across the genome. TthPrimPol synthesizes the DNA primers needed for Phi29 DNA pol, allowing for the exponential amplification of genomic DNA without primer artefacts. Analyses on genomic DNA amplified from single Hek293 cells in comparison to non-amplified DNA reveal superior genome coverage with little bias, excellent SNV recovery and minimal introduction of error such as allelic dropout or chimera formation.
Originally aired 29 October 2015, GenomeWeb
Presented by Mark Liles, Auburn University, Bob Klein, USDA, and David Smith, Mayo Clinic.
This webinar will focus on a range of research and clinical applications enabled by improvements in mate pair technology for whole genome sequencing.
Speakers will discuss their use of Lucigen's NxSeq long mate pair library kit for a number of applications, including closing and finishing microbial genomes, resolving complex plant organelle genomes, and characterizing genetic alterations in cancer.
Mark Liles, associate professor at Auburn University, will describe his use of the technology to finish multiple bacterial genomes using a single 10-20 kb mate pair library in conjunction with a conventional 600 bp paired end fragment library. His presentation will address the implications of this approach for assembling repeat-rich, complex genomes from fungi, mitochondria, chloroplasts, plants, and animals.
Bob Klein, research geneticist at the US Department of Agriculture's Agricultural Research Service, will share details of a project to sequence and assemble the mitochondrial and chloroplast genomes of sorghum. De novo sequence assembly of these large, repeat-rich interrelated genomes is complicated by significant lateral gene transfer between the organelles, but his team was able to use a new long span mate pair library construction strategy to unambiguously assemble both genomes.
Finally, David Smith, professor of laboratory medicine and pathology at the Mayo Clinic, will discuss the use of next-generation sequencing in cancer management In particular, he will focus on the use of mate pair sequencing to characterize genomic alterations in an individual cancer and how this knowledge can be used to both monitor the cancer through treatment regimens and to develop the most appropriate clinical regimen for that cancer.
Originally aired 19 February 2015, GenomeWeb
Presented by Richard Davis, Auburn University, Miroslav Valarik, Institute of Experimental Botany, and David Mead, Ph.D., Founder, Lucigen.
This online seminar describes new technology capable of generating long range sequencing information, enabling de novo genome assembly, closure, and finishing on the Illumina or Ion Torrent next-generation sequencing platforms.
Despite advances in NGS technology, true closure and finishing of genomes or bacterial artificial chromosomes remains extremely difficult. NGS instruments produce gigabases per run, but the short read lengths and small size of sequenced fragments result in gaps, misassembled contigs, collapsed repeats, and missing sequences, leaving these regions to be finished manually, if at all.
With new long span NGS read technology, however, user-defined 2-8 kb mate-pair or 10-20 kb mate-pair libraries can be produced with bead-based or gel purification protocols, with the potential for up to 100 kb mate-pair libraries in the future. Featured speakers will highlight real-world applications of this technology and the utility of long span mate pair libraries for genome closure.