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Pushing the Size and Input Limits of DNA Fragment Library Prep and Streamlining NGS Library Prep with Automation

Wednesday | July 11, 2018

8 AM CST (9 Eastern, 6 am Pacific) Register Now
11 AM CST (Noon Eastern, 9 am Pacific)  Register Now

Pushing the Limits of Next Gen Sequencing: Libraries from the Ultra-limited and the Ultra-small

As next gen DNA sequencing popularity grows, researchers continue to push the limits of current technologies to accommodate challenging sample types and novel applications. To make these experiments possible, multiple steps in the NGS library prep workflow must be optimized for both amplification-free and PCR based libraries. This presentation will examine some of the key factors involved in building high quality libraries from limited sample inputs as low as 50 pg, and from ultra-small fragments under 100 bp. Data from cell-free DNA and ChIP inputs as well as tiny synthetic oligos will be presented.

Solving Sample Prep Challenges Through Automation

Consistency and reliability are areas where automated methods, especially in liquid handling, can greatly enhance results when compared to manual methods. NGS sample prep is one example of a laboratory process where a robotic pipettor provides significant benefits vs. manual sample prep. Automated pipetting, following carefully prepared scripts, improves the consistency of sample handling, reliability of method performance and insures accuracy of data tracking and recording. This presentation will provide specific examples of how automated pipetting and control of the process will improve these factors and result in greater sample throughput and better data.

Scott Monsma

Presented by
Scott Monsma, Ph.D.
Director, NGS Services,

Lucigen

Phil Farrelly

Presented by
Philip Farrelly, B.S. & M.B.A.
CEO, Hudson Robotics


Webinar IconRecorded Webinars

CRISPR 101: Optimizing Your Gene Editing Experiments

Aired Wednesday, 9 May 2018
Michele Auldrige, Senior Scientist, Ph. D. and Beth Frey, Product Manager, Lucigen

CRISPR is a revolutionary technology that has advanced many areas of research around the world and is moving at a breakneck pace. Understanding the basic challenges of CRISPR gene editing and how to overcome them is essential to successful use of CRISPR in new applications. In this webinar we will examine the use of CRISPR Cas9 Ribonucleotide protein (RNP) complexes, discuss basic gene editing methods, and provide helpful hints for impactful experimental designs. We’ll review options for guide RNA (gRNA) generation, best practices in RNP formation and delivery, as well as in screening and detection of edited cell populations. Finally, we’ll provide examples from the literature and review tips and tricks to help you get the most out of your gene editing experiments.

Key Learning Objectives:

  1. How to choose the best guide RNA generation methods for your application
  2. When to choose DNA vs RNP gene delivery for different research goals
  3. How to achieve clear, decisive results from your T7E1 mismatch detection assays

Download the Slide Deck


Loop-Mediated Isothermal Amplification (LAMP): Primer Design and Assay Optimization

Aired Wednesday, 14 March 2018
Tony Rockweiler, Diagnostic Research Scientist 2, Lucigen

Nucleic acid based detection assays hold great promise for improved speed and sensitivity for a variety of applications such as microorganism detection, disease diagnosis and more. Loop-mediated isothermal amplification (LAMP), an isothermal nucleic acid amplification method which is rapidly gaining popularity, provides assay developers with a fast and cost-effective alternative to PCR for nucleic acid detection. LAMP’s isothermal characteristics allow for the development of simple, robust, low cost assays that can be deployed for point-of-care, point-of-need or environmental testing. Although a developed LAMP assay provides robust detection capabilities, initial design and development can prove difficult due to the 4 (or 6 for the faster assays) primer requirement. The need for 6 primers targeting 8 regions of the genome, the distance requirements between the primers, and sequence variability among clinical isolates of a given species put significant constraints on primer design. Improper primer design or selection leads to suboptimal assays resulting in slow amplification, non-specific amplification, and poor sensitivity. This webinar will review the basic mechanics of LAMP and how it may be used as a diagnostic assay. We will look in-depth at primer design with a focus on the use of primer design software and how specific settings improve primer design success specifically with LavaLAMP™ Enzyme-based Kits. We will also discuss optimization techniques to get the most out of your assay design.

This webinar will:

  • Review the mechanics of LAMP
  • Examine the primers required and their design
  • Learn how adjust software setting for better primer design
  • Demonstrate the effects of optimizing primer design on LAMP assay results
  • Explore additional ways to optimize LAMP assays

Download the Slide Deck


Dirty DNA is Okay - Sometimes! Obtaining Surefire and Rapid PCR/RT-PCR Results from Diverse Sample Types

Aired Wednesday, 24 January 2018
Karen Kleman, Ph. D. Product Manager, Lucigen

Whether you are confirming genomic alterations in transgenic mice or screening CRISPR-edited clones using T71E1 assays, sample preparation and PCR analysis can take a lot of time. It’s important to optimize each step of your assay to achieve an efficient workflow, reproducible results and reliable data. In this webinar we will review each step of end-point PCR/RT-PCR assays including (i) an investigation into potential contaminants in your sample, (ii) choosing the proper DNA or RNA extraction method, and (iii) identifying the best fitting amplification enzyme or master mix. We’ll also explore how to overcome assay development challenges in your research and discuss solutions that increase efficiencies in popular end-point PCR/RT-PCR applications.

Key Learning Objectives:

  • Discuss when to use extracted vs. purified nucleic acids in various applications
  • Identify the potential stumbling blocks in the PCR and RT-PCR workflows
  • Explore the utility of fool-proof PCR systems for all template types
  • Provide examples of quick and effective PCR screening applications

Download the Slide Deck


Lowering Next Gen Sequencing DNA Input Requirements and Gaining Access to More Samples

Aired Wednesday, 8 November 2017
Rob Brazas, Ph. D., Senior Product Manager, Lucigen

Next gen sequencing (NGS) is rapidily growing in popularity due to the power of the technology. As its popularity increases, researchers continue to push the limits of current technologies to accommodate challenging samples and applications. In order to access these difficult sample types and applications, multiple steps in the NGS workflow must be optimized including DNA fragment library preparation and PCR amplification. This webinar will look in-depth at library preparation with a special focus on the key steps of adaptor ligation and PCR amplification. We will explore how both maximizing efficiency of adaptor ligation and increasing uniformity of PCR amplification across all DNA library fragments can minimize PCR bias and increase sequencing results quality. Specifically, we will introduce the new NxSeq® UltraLow DNA Library Kit which meets these important requirements, enabling the production of high quality DNA fragment libraries from as little as 50 pg to as much as 75 ng of DNA.

This webinar will:

  • Review DNA fragment library construction
  • Discuss sequencing duplicates and the role of library input amounts and sequencer capacity
  • Introduce the NxSeq® UltraLow DNA Library Kit
    • Library construction mechanics
    • Analysis of adaptor ligation efficiency
    • Whole genome sequencing results from MiSeq, HiSeq 2500 and HiSeq X Ten Instruments

Download the Slide Deck


Leveraging the Power of In Vitro Transcription: An Exploration of Applications, Challenges and Solutions

Aired Wednesday, 13 September 2017
Rob Brazas, Ph. D., Senior Product Manager, Lucigen

RNA was long underrated as simply the cellular messenger of genetic information between DNA and expressed proteins. It’s now clear that RNA molecules play many roles within cells in addition to translation (mRNA), such as translation inhibition (miRNA), bacterial defense (CRISPR gRNA) and as transmission of genetic material for many viruses.

In vitro transcribed RNA is an integral component in many research projects aimed at understanding these important cellular functions and viruses. The speed and scale of RNA production, as well as the ability to incorporate chemically modified nucleotides, support an array of exciting research applications.

This webinar will:

  1. Review in vitro transcription (IVT) technology
  2. Summarize applications of IVT RNA including examples from the literature
  3. Discuss some of the key challenges of IVT
  4. Provide potential solutions to those challenges including the AmpliScribe™ T7-Flash and DuraScribe® T7 Transcription Kits

Download the Leveraging the Power of In Vitro Transcription Slide Deck


Accelerate your Research with Synthetic DNA and Hands-free Cloning

Aired Wednesday, 12 July 2017
Julie K. Robinson, M.B.A., Senior Product Manager, Synthetic Genomics, Inc.

Artificial gene synthesis (DNA printing) is a powerful synthetic biology tool allowing fast, error-free assembly of almost any desired DNA sequence. Many modern genomics applications start with gene synthesis, including protein production, antibody library generation and cell engineering. In this webinar we will discuss how you can take control of your workflow and accelerate your research by synthetically building and cloning genes and gene variants on your own benchtop. We’ll learn how the BioXp™ 3200 System assembles your gene of interest in silico, utilizing specific and targeted sequence modifications for more efficient results. We will discover how this versatile genomic workstation creates your gene, automates cloning and advances the DNA through various workflows to streamline and speed genomic discovery.

Key Learning Objectives

  • Learn about the power of Synthetic Biology
  • Answer key questions about gene function with variant synthesis
  • Explore new cloning and genomics workflows to accelerate your research
  • Review new genomic applications like automated Next Gen Sequencing Library Construction

Download the Accelerate Your Research with Synthetic DNA and Hands-free Cloning Slide Deck

SGI-DNA, Synthetic Genomics, IncorporatedThis webinar is brought to you in collaboration with SGI-DNA providers of genomic solutions to advance scientific discovery. SGI-DNA’s ever expanding suite of products, services, reagents, bioinformatics tools and instrumentation enables scientists to discover, design and build novel solutions for basic research, as well as for biomedical and industrial applications.


Increase Your Probability of Success with Faster, Easier Protein Expression in E. coli

Aired Wednesday, 10 May 2017
Beth Frey, Product Manager, Lucigen

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.

Key Learning Objectives:

  • Discover methods to simplify protein expression in E. coli for single or multiple targets
  • Learn how highly tunable promoters can help overcome protein toxicity
  • Explore examples of novel fusion tags that enhance expression and solubility
  • Discuss best practices for efficient fusion tag removal

Download the Increase Your Probability of Success with Faster, Easier Protein Expression in E. coli Slide Deck


How Can Transposons Accelerate Your Genomic Research?

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

  • Learn how transposons randomly insert into DNA
  • Discover how transposons can speed your results for multiple microbial genetics applications
  • Explore the differences between in vitro and in vivo applications
  • Learn how to design and use your own transposable sequences
  • Maximize your success with tips and tricks from the experts

* These Epicentre-branded kits are now exclusively available through Lucigen.

Download the How Transposons Accelerate Your Genomic Research Slide Deck


Whole Genome Amplification (WGA): What to Do When You Don’t Have Enough Genomic DNA

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

  • Learn the basics of multiple displacement amplification (MDA) for whole genome amplification
  • Explore the different forms of MDA kits available
  • Investigate the performance differences of the various MDA kits 
  • Look in depth at the use of whole genome amplification followed by next gen sequencing

Download the Whole Genome Amplification (WGA): What to Do When You Don’t Have Enough Genomic DNA Slide Deck


Loop-Mediated Isothermal Amplification (LAMP): Assay Development Challenges and Solutions

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.

Key Learning Objectives

  • Learn basic design of LAMP assays and the key differences from PCR

  • Learn key parameters that affect assay performance and approaches to assay optimization

  • Explore the common causes of assay failure and how to troubleshoot

  • Discover ways to apply LAMP in point of care/point of collection using a novel thermo-tolerant LAMP master mix that is room temperature stable when freeze dried

Download the Loop-Mediated Isothermal Amplification (LAMP): Assay Development Challenges and Solutions Slide Deck


Strategies for Improving Soluble Protein Production in E. coli

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.

Download the Strategies for Improving Soluble Protein Production Slide Deck


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.

Agenda:

  • Selecting your competent cell format:
    • Electrocompetent versus chemically competent
  • Maximizing transformation efficiency to create large libraries
  • Choose the right strain:
    • Strain genotypes and properties
    • Specialized strains for specific applications
  • Engineered strains for low-endotoxin protein and plasmid preparation

Download the Competent Cells 101: Maximizing Your Success in Cloning, Protein Expression, and Library Generation slide deck


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.

Agenda:
This webinar will cover the following topics regarding cloning difficult DNA targets:

  • Characteristics that make DNA difficult to clone
  • Vector technologies that help
  • Difficult cloning data examples
  • Ligase-independent cloning
  • E. coli strains engineered to tolerate DNA encoding toxic proteins
  • Summary

Download the Clone the Unclonable - Vectors and Cells to Capture and Express Problematic DNAs slide deck


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.

Download the Next Gen Sequencing: Library Prep Challenges and Solutions slide deck


Eliminate Bias in Single Cell Whole Genome Amplification with the TruePrime™ System

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.

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