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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.
If you are a Michelin star chef, then your first priority for preparing your signature dish is to use the best ingredients. One rotten potato or one slightly overripe strawberry could ruin not just a dish, but also your reputation. In the laboratory we are (mostly) not cooking rotten potatoes, but we are doing delicate…
As we discussed previously, the gaps in our understanding of the human genome make variant classification an extremely difficult job. However, with each passing day our knowledge increases, and the tools to help us become increasingly more efficient. Let’s pick up where we left off in our first article about variants. After checking Ensemble to…
Imagine that you want to test the efficiency of an antimicrobial treatment in inhibiting a certain bacterial pathogen. As part of the experiment, you expose the bacteria to the treatment and monitor the cultivability of the microorganism by counting the number of colony forming units (CFU) formed on culture media. If the microorganism is sensitive…
Negative staining of proteins is a versatile tool for structural biology. The sample preparation protocol is simple: the sample is embedded in a heavy metal stain that gives rise to increased specimen contrast. Thus, negative staining is a very convenient method to assess sample homogeneity, formation of macromolecular complexes, or quality of protein preparation. Conventional…
Have you ever seen a mouse chowing down on its dinner and wondered how it translates to energy? Well, that’s exactly what is keeping some scientists up at night, and luckily these questions are now becoming easier to answer in quite significant detail. Over the last couple of decades, several companies have developed ‘metabolic cages’….
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…
Chromatin immunoprecipitation sequencing, better known as ChIP-seq, is a massively parallel approach for understanding the interactions between proteins and DNA. This is especially important for determining the activity of transcription factors, which is why it’s frequently used to learn about the complicated series of biological steps leading to cancer. It’s also key to many epigenetic…
It’s Sunday morning, the sun has just begun to rise, and you find yourself on the way to the lab (again!), sipping hot coffee and melancholically thinking of your abandoned bed. But something is different this time. Today, the freezing-cold wind blowing from behind is not the only motivation pushing you to sacrifice another weekend in…
Fourier transform infrared spectroscopy (FTIR) is an extremely sensitive technique for measuring the absorption and intensity of electromagnetic radiation in the infrared region of the spectrum of either a solid, liquid or gas sample. You can use FTIR to: How Does Fourier Transform Infrared Spectroscopy Work? Briefly, FTIR measures how well a sample can absorb…
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…
Just like you need to clean up your room from time to time, your cells also need to do a bit of housekeeping. Your cells accomplish this through a process called autophagy. Autophagy mainly serves two roles. The first is to remove damaging materials, such as misfolded proteins, dysfunctional organelles, and foreign invaders. The second…
Because of the ease of performing controlled crosses, maize (or corn (Zea mays)), has been a staple of plant genetics research for decades. Barbara McClintock herself chose maize as her research organism for her Nobel Prize winning work. If you are looking to get involved but aren’t sure how to get good yields in the…
You may have heard about a breakthrough cancer therapy that engineers patient’s immune cells to fight their cancer using chimeric antigen receptor (CAR)-T cells. If you don’t live in the world of immunology, you may not know what a CAR is, or what it is used for. Here you’ll find a brief guide to CARs,…
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…
What Is Optical Mapping and How Is It Used? Synchronisation of the contraction of heart muscle is essential for the efficient pumping of blood through the circulatory system. Cardiac contraction is controlled by the regulated spread of electrical impulses from cell-to-cell within the heart. In pathological conditions, these electrical impulses can become disordered and lead…
RNAscope is a new method of quantitative RNA in situ-hybridization that has taken laboratories by storm. Learn advantages over traditional techniques and how RNAscope works in this introduction article.
The decreasing costs in genomic sequencing over the past decade have inspired researchers to apply shotgun next-generation sequencing to entire microbial communities. While the reads generated typically cannot be assembled cleanly into individual genomes, there is often enough information produced to identify most microbes present in the population. However, this approach lacks sufficient resolution to…
Researchers have relied on immunodetection techniques such as Western blotting, flow cytometry and Enzyme-Linked Immunosorbent Assay (ELISA) for years, but immuno-PCR is a relatively new method. By merging an ELISA with the Polymerase Chain Reaction (PCR), immuno-PCR provides extremely high levels of assay sensitivity. ELISA An ELISA is an assay in which a molecule is…
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…
There is something about kinases that resemble ghosts. Their effects reveal their presence, but they can be difficult to catch. With a low abundance of hundreds or even tens of molecules per cell, they are difficult to detect using conventional methods such as Western blotting or mass spectrometry (MS). However, you will need to detect…
Since our early steps in Science we have been told that every cell in our body has the exact same genetic information (minus one or two alterations). Therefore, the great variety of cells in our body comes from gene expression – each cell must express one set of genes and repress another set to function…
In any application that uses antibodies for signal detection (e.g., Western blotting, ELISA, immunohistochemistry, or FACS), there are two approaches to antibody labeling: direct and indirect labeling. Standard Western blotting uses indirect labeling because you use a primary antibody to detect the target antigen, followed by a secondary antibody to which a detection molecule is…
Proteins in the cell are in a constant flux governed by events including synthesis and degradation. In an effort to make cells more efficient by reducing the unnecessary protein load, most proteins in the cell have a specifically defined half-life. Another reason why cells have evolved to degrade proteins is to ensure timely removal of…
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…
Target capture through PCR has been a mainstay in genomics for years, but scientists working on especially repetitive, poorly characterized, or rapidly evolving regions continue to struggle to fish out those stretches of DNA for further study. However, whole genome sequencing, the only other alternative for these regions, can force researchers to pay for much…
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,…
If you have studied cellular movement or cell division, you have encountered Rho in the literature, because it regulates both processes. And the list of roles for Rho in the cell continues to grow! The prominence of Rho in the biology of non-diseased and diseased cells has caused researchers to continually optimize the Rho pull-down…
If you have ever had a home garden, you are probably familiar with the fact that adding a little fertilizer to a plant can really do wonders. This can also be the case in a lab greenhouse! The difference is that instead of adding a bit of the “blue stuff,” we try to be a…
The primers for your gene of interest have finally arrived in the mail, and you’re ready to figure out whether your favorite gene’s expression level is elevated in those precious tissue samples. Only one small last step before you can proceed. Primer validation. This is a standard procedure where you run PCR or qPCR on…
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…
Interest in the three-dimensional structure of chromatin has exploded over the past few years—and for good reason! We now know that DNA isn’t randomly piled into the nucleus like a bowl of spaghetti, but arranged in functional loops and domains, more like a city blueprint. This is particularly exciting to (epi)genomic scientists, because 3D chromatin…
Automated image analysis uses finely tuned software to extract data from digital images. Algorithms recognize specific shapes and patterns in the images and gather quantitative information that is then used for further data analysis. The pharmaceutical and biological research industries have benefitted greatly from this technology, which allows researchers to analyze hundreds—if not thousands—of samples…
Thinking about studying extracellular RNA and in particular microRNA? Read on for some great tips for extracting circulating microRNA.
As an undergraduate student, one of the first experiments I did was staining chromosomes in mitotically active onion root tip cells. The stains that are conventionally used for this purpose are acetocarmine or aceto-orcein (which smell like vinegar). However, the cost of these stains is quite high. Personally, I find safranin, which is another stain, more…
Kidney Modeling with Kidney Organoids Derived from Human Pluripotent Stem CellsStem cells are a valuable tool for kidney disease modeling as well as experimental regenerative medicine and drug screening. There are more than twenty different cell types in the mature kidney, which adds to the complexity of the model, but also provides the opportunity to…
Is ELISA giving you the blues? The frustrating kind, not the lovely kind you get while watching the enzyme substrate reaction! This age old assay has the perks of being quick and fairly simple to perform, but when conditions are not perfect, ELISAs can deliver less than optimal results, and fail to be consistent and…
Welcome back, fellow flow cytometry friend! I am sure that you are rocking your data acquisition at this point, having perfected your understanding of panel setup, fluorophore usage, and using the flow cytometer of your choice. However, with as many samples as you are running, it is possible that you may be experiencing a little…
My favorite reason for being a biologist is that I am endlessly amazed by how life adapts to various pressures on planet Earth. This especially holds true for endospores, one of nature’s most resilient means of surviving for thousands of years in non-ideal environmental conditions. In this article, we’ll explore some of the extreme environments…
The technology for DNA sequencing was developed back in 1977 thanks to Frederick Sanger. It took a bit longer before it was possible to sequence a complete genome. This is because we needed an appropriate mathematical model and massive computational power to assemble millions or billions of small reads to a larger complete genome. Today’s…
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