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DNA / RNA Manipulation and Analysis

Show Your Molecular TALEN(T)

Introduction Did you know that the idea of using genetic engineering to ameliorate certain human diseases was viewed as ‘science fiction’ only 10 short years ago? While cell mutagenesis studies and genetic knockout experiments were feasible before genetic engineering, they were not very reliable. Indeed, due to the random and imprecise nature of these older…

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NGS Target Enrichment Strategies

Next-generation sequencing (NGS) has ushered in a new era of understanding of both the inner workings and the function of the genome. NGS allows researchers to look at traits—including diseases—that are linked to differences or mutations in an individual’s genes. Since only about 1% of the human genome constitutes genes that code for proteins, several…

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The EMSA – Teaching an Old Dog New Tricks

Probing Nucleic Acid-Protein Interactions with EMSA The electrophoretic mobility shift assay (EMSA) is a powerful technique for detecting specific-binding of nucleic acid-protein complexes. Over the past 30 years, EMSA has been the “go to assay” to investigate the qualitative interactions between nucleic acids (DNA or RNA) and nucleic-acid binding proteins. Through the use of radio-labeled…

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Isolating Bacterial RNA from Blood

For many decades, the only way to detect sepsis – bacterial growth in blood – was isolating the bacteria and growing bacterial colonies on a special medium. This was done by first spinning down the blood, which brought the blood cells and bacteria into the pellet. The pellet was spread on a blood agar plate…

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How (and Why) to Label Nucleic Acids

Have you ever wished you could snag individual strands of DNA or RNA with a lasso? Or look at them one by one, figuring out exactly where they are or what they are doing? Fortunately, there are techniques that exist to label nucleic acids for their visualization and purification! Nucleic acids can be labeled at…

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The Beginner’s Guide to Reading Plasmid Maps

Very often plasmid maps, especially historical ones that are hand-drawn by a long-forgotten PhD student, are a puzzle. What are these arrows and boxes? Where do I start? Don’t worry, we have a crash course introduction into deciphering plasmid maps. Familiarizing Yourself with Your Plasmid of Interest Let’s start with a classic plasmid: pBR3221. It…

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RNAseq Library Preparation: From Cells to cDNA

RNAseq libraries, also called whole transcriptome shotgun sequencing libraries, provide a snapshot of cellular processes. This allows the researcher to gain information regarding changes in transcriptome in response to environmental changes, during disease, or after a drug application. RNAseq libraries also allow for the detection of mRNA splicing variants and SNPs. RNAseq libraries have virtually…

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Picking the Right DNA Isolation Kit for Your Application

If you plan to work with purified DNA in the lab, it’s likely that you will use a commercial DNA extraction kit to isolate and purify your DNA of interest. With so many types of kits available, it can be a major challenge to choose the best one to use when working with an unfamiliar…

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Are You In(to) Situ? – Putting Together Your First RNAscope® Assay

You are thinking of trying out RNAscope®. After all, RNAscope® holds promise for increasing the sensitivity and specificity of your in situ hybridization. Yet, getting started can be a little overwhelming with the numerous kits and reagents available in the RNAscope product line. Here’s an overview of your options to help you navigate to the…

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RNA Strandedness: A Road Travelled In Both Directions

For most molecular biology purposes, DNA is thought of as a string of nucleotides running from 3’ to 5’, and the corresponding mRNA sequence is complementary to this DNA string. However, visualizing this quirky DNA structure for what it is – two antiparallel strands joined together – it quite important for many applications, such as…

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The Next Big Thing: Alternative Polyadenylation

What Is Alternative Polyadenylation? Processing of mRNA and its regulation plays a fundamental role in gene expression. As science progresses, alternative polyadenylation takes center stage in the undercurrents of gene expression. 1,2 Polyadenylation is part of the pre-mRNA maturation process and involves polyadenylation of the 3’ end of the emerging RNA.  This process happens to…

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The Importance of Non-coding RNAs

What Are Non-Coding RNAs? What was once considered “junk” may end up being the most important part of our genome. Non-coding RNA (ncRNA) is RNA that is transcribed from DNA but diverts from the “central dogma” because it does not code for proteins. NcRNAs are ubiquitous in eukaryotes: while 90 percent of eukaryotic genomes are…

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Protocols for Cloning Without Restriction Enzymes or Ligases

There are many cloning methods that do not require restriction enzymes or ligases. Read below to learn about how to achieve seamless cloning results via Topoisomerase cloning, SLIC, and Gibson. Method #1: Topoisomerase Technology Topoisomerase technology requires no special primers and no ligases – it is as easy as cloning comes. This technology is based…

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Cloning Methods: 5 Different Ways to Assemble

Over the past few decades molecular biologists have developed procedures to simplify and standardize cloning processes, allowing vast arrays of artificial DNA structures to be more easily assembled. Are you familiar with all the cloning options out there? Let’s look at five different cloning methods you can use to get your construct. At the end…

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Ligation Independent Cloning Primer Design

Ligation independent cloning (LIC) is an easy and effective method to ensure successful cloning, all without the need for ligation. As easy as the technique is, designing primers can be a bit tricky. In this article, we will present a quick overview on primer design for ligation independent cloning.

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Nucleic acids 101: Confirming Their Quality

Nucleic acids 101: Confirming Their Quality Join us in this webinar as Dr. Victoria Doronina helps you determine the quality of your nucleic acids. In this webinar you will learn:In this tutorial, you will find: How to choose the best method to extract your nucleic acids Which method you should chose to determine nucleic acid…

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A Beginner’s Guide to Lentiviral Transduction

The use of viral delivery systems to transduce cells for gene and protein investigations has become prominent over the last 20 years. In particular, the use of lentiviral vectors permits stable expression of your gene of interest. This is all possible with a little bit of nucleic acid magic. Lentiviruses (a genus of retrovirus) express reverse…

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How to Use CRISPR to Accelerate Cancer Therapies

How to Use CRISPR to Accelerate Cancer Therapies Join Theo Roth as he describes his lab’s novel CRISPR-Cas9 genome-targeting system that does not require viral vectors to modify T cell genomes, but instead focuses on HDR. This allows rapid and efficient insertion of large DNA sequences at specific sites in the genomes of primary human…

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Using CRISPR/Cas9 to detect sequences on single DNA molecules with high-speed AFM

Using CRISPR/Cas9 to detect sequences on single DNA molecules with high-speed AFM Join Dr. Jason Reed as he describes a novel method by which endonuclease-inhibited Cas9 can be employed as a programmable biomarker in high-speed atomic force microscopy (HS-AFM) imaging.In this tutorial, you will find: How CRISPR/Cas9 can be used to “flag” alterations and mutations…

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Old Reliable: Two-Step Allelic Exchange

Manipulating the genes of organisms is crucial for studying their functions. In times before genetic engineering, scientists would shoot bacteria with X-rays or expose them to destructive chemicals until spontaneous mutations would arise. Fortunately, current methods are more sophisticated and less torturous. Researchers now use more directed techniques to introduce mutations. There are several ways…

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A Start to Finish Guide to Target Gene Validation Using Quantitative RT-PCR

A Start to Finish Guide to Target Gene Validation Using Quantitative RT-PCR Speaker Matthew Mule In this tutorial, you will find: While next generation sequencing enables researchers to unveil expression levels of the entire genome, qRT-PCR remains the gold standard for measuring transcript levels of individual genes for functional studies and for the purposes of…

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The Use of qPCR to Validate Epigenetic Enrichment of Pathogen DNA from Complex Samples and Human DNA from Stool

qPCR is one of the most specific and sensitive tools in molecular biology, allowing the quantification of target DNA molecules present at less than 1 in 106. Next Generation Sequencing (NGS) has similar potential. However, the presence of large amounts of non-target DNA in most clinical or environmental samples precludes easy and inexpensive analysis of…

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RNA Isolation from Drosophila – Don’t Let the Cuticle Scare You!

Isolating RNA from either Drosophila larvae or adult heads can seem a bit daunting, primarily due to the presence of the cuticle. The cuticle is a protective exoskeleton comprised of insoluble collagens, cuticlins, glycoproteins, and lipids. While it may take some force to remove the cuticle, you can do this easily and without compromising your…

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A Guide to Harnessing DNA Assembly for Drug Discovery

Current DNA synthesis and assembly technologies give today’s genetic engineers unprecedented freedom to control every aspect of genetic design. In this webinar on DNA assembly, you will learn: Key concepts in DNA assembly The importance of analyzing combinatorial libraries of genetic designs for natural product biosynthesis Emerging areas of research Join Dr. Michael Smanski as…

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Small Differences that Matter: Detecting Microsatellite Polymorphisms

If you have any training in genetics, chances are that during the course of your education you ran into those funny little sequences called microsatellites. These are repeated tandem motifs 1-6 nucleotides long, scattered all over our genomes. These used to be called “junk DNA,” because researchers thought that the repeats served no purpose. Nowadays,…

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DNA Footprinting

Studying DNA–protein interactions is an important aspect of molecular biology. Researches use a variety of methods to study these like the chromatin immunoprecipitation (ChIP) assay, electrophoretic mobility shift assay (EMSA), DNA pull down assay, luciferase reporter assay, filter binding assay, yeast one hybrid system, etc. Another interesting assay that helps investigate DNA–protein interactions is the…

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Bacterial Transformation Troubleshooting for Beginners

The first time I did a transformation was when I worked with site directed mutagenesis. I cloned a protein sequence into the p15TVL vector, created my mutants (but that’s another story), and was finally ready for the next step: transformation and expression of my desired protein. Little did I know that my enthusiasm would fall…

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Restriction Enzymes: Five Things to Consider Before you Chop!

The use of restriction enzymes to characterize DNA has been popular since the 1970s. Today, this “old school” technique is still one of the easiest and fastest ways to assess DNA sequences. Like most lab reagents, restriction enzymes can be fickle and you should bear a few things in mind when using them. Generally, sticky-ended enzymes have greater…

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Those Site-Specific Recombinases in Your Tool Kit

Most of us are aware of genetic engineering systems like Cre-Lox, TALENs, Zinc finger systems, and of course, CRISPR-Cas9.  These are all examples of CSSR- Conservative Site-Specific Recombination. We use these site specific recombinases routinely, but do we really know about them or what the future hold for these tools? It turns out that CSSR…

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Using Synthetic DNA For Long Term Data Storage

The amount of data requiring long-term storage is growing and accelerating. Current long-term digital storage technology cannot keep up. Imagine roughly 2.5 QUINTILLION bytes of data being created everyday in this world1–2 as more computers and network infrastructure come online. For average users, a long-term storage solution is probably not an issue. However, organizations and…

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FISHing for miRNAs in Archived Tissues? Yes, It Is Possible!

We use fluorescent in situ hybridization (FISH) techniques routinely to detect DNA or RNA sequences in tissues, but what about micro RNAs (miRNAs)? No worries, FISH is now optimized to meet the challenge. To help you get going with the method, here’s what you need to know. The first thing that comes to mind when…

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Transfection Toolkit

Engineering a mutation or overexpressing a recombinant protein to study and characterize its function in mammalian cells is no easy task. Luckily, Chinese hamster ovary (CHO) cells, which have been a mainstay in the lab since the 1950s, represent a relatively easy mammalian model system to engineer. There are several methods to choose choose from…

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Get Your Polymerase Cycling Assembly Oligos Together

The polymerase chain reaction (PCR) is the backbone of many lab techniques. In short, it allows for the exponential amplification of a specific segment of DNA. Through the use of primers encoding restriction enzyme sites, these amplified fragments are used in downstream cloning procedures, usually leading to the insertion of one, maybe two, PCR fragments…

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How to Perform DNA Extraction from Dried Blood Spots Using Chelex Resin

Every bio- scientist who wants to analyze DNA knows that the process begins with the extraction of DNA from cells of interest. These cells could be RBCs, parasites, or bacteria to name a few. Furthermore, there are various DNA extraction methods1  to choose from depending on sample type, downstream analysis, and so forth. Many scientists…

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Benzyl Isoamyl Alcohol: a Novel, Bizarre, and Effective DNA Purification Tool

DNA Purification We all use our favorite techniques for DNA cloning, such as Gibson assembly, TOPO cloning, ligation independent cloning (LIC), and TA cloning. However, DNA purification methods themselves, haven’t changed all that much since the 90’s. Historically, the introduction of phenol extraction in 1956, to purify nucleic acids from rat liver, rapidly replaced previous…

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Guide to CRISPR/Cas9 Delivery: How to Maximize Your Editing Efficiency

In this webinar, you will learn how to maximize your genome editing efficiency using CRISPR/Cas9 and how to apply this technique in your research. The main points in the webinar will include: How to design guide RNAs using online tools specific to the genome and application of interest. Tips and practical advice to assist you…

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Quantifying & Assessing RNA: TE or not TE?

Red Pill or Blue? Carrying out science often involves many difficult decisions! I see it all the time in RNA protocols – the “gracious” option of using purified water or Tris-EDTA (TE) buffer to dissolve (or elute, if you are using column purification) RNA. When I was trained in assessing RNA using UV spectrophotometry, graduate…

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