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DNA / RNA Manipulation and Analysis

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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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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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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4 Important Considerations for Your Cell Lysis

You’ve cultured your cells and completed your treatments, now it’s time to harvest them and proceed to the downstream effects. Cell lysis is the crucial stage that determines if your experiment has a chance of producing the data that you have been waiting for. Part of the starting biological material is inevitably lost on each…

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New-ISH on the Block: Introduction to RNAscope®

When sensitive detection of RNA is required, many scientists turn to qPCR as it is a versatile technique that can detect many different types of RNAs from mRNA, non-coding RNA, to microRNA. However, if you also require spatial information, like which cells are producing your RNA, the technology of choice has historically been in situ…

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Get to Know Your Reference Genome (GRCh37 vs GRCh38)

Whether your experiment relies upon a reference-based genome assembly or mapping reads to a reference genome to identify variants, you need to choose a human reference genome assembly. But wait! You go to the FTP site of NCBI’s refseq and click on the Homo sapiens folder. There you are presented with two choices. Which one…

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Extracting Circulating microRNA, Part Two: Isolation Methods

In the second part of this two part series on analyzing extracellular microRNA (miRNA) from blood or serum, we continue with the methods for miRNA extraction. Generally, there are two different approaches for microRNA isolation. The first of these is organic, liquid-liquid. The total RNA extraction method employs phenol/chloroform isolation with guanidine isotiocyanate as a…

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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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Extracting Circulating microRNA, Part One: The Preanalytical Phase

microRNA (miRNA) is leading the way as circulating biomarkers in disease biomarker discovery. The hype around circulating miRNA is warranted. They meet many criteria for a good biomarker—namely, stability in circulation and varying levels in certain pathological conditions. Recently, studies have shown that miRNAs could be useful markers for cancer, myocardial infarction, atherosclerosis, and neuro-degenerative…

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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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How UV Radiation Causes DNA Mutations

We all know that we are supposed to put on sunscreen in the summer months to protect ourselves from skin cancer, and the connection between sun exposure and cancer is well documented (Koh et al., 1996; Armstrong and Cust, 2017). UV-A and UV-B rays from the sun interact with the DNA in our skin and…

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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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DNA Extraction from FFPE Tissues for NextGen Sequencing

Rapid genomic analysis offered by next generation sequencing (NGS) is ideal for personalized medicine approaches to clinical genetics, microbiological profiling, and diagnostic oncology. Many standard clinical samples are preserved as formalin-fixed, paraffin-embedded (FFPE) tissues, which presents obstacles for use in NGS analysis. FFPE tissue preservation has the benefit of keeping samples intact for histological examination…

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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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