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It’s unclear exactly how the term ‘Special Stains’ first arose in the world of histology, but it refers to empirical and histochemical staining techniques that significantly contributed to the advancement of histology in the late 19th century. In a nutshell, these stains are ‘Special’ because they are not routine – simple as that. Therefore, Special…
Mycoplasmas are the most difficult-to-detect organisms in your eukaryotic cell culture. And they can be the most dangerous; they can disrupt cell growth and differentiation and even apoptotic patterns without you even knowing what’s going on until it’s too late. Traditional cell culture methods can take up to a month to yield results, which means…
In a typical biology lab, you may encounter many types of biological materials, including cells, bodily fluids, purified DNA and RNA, enzymes, bacterial cultures, body parts, and whole animals. In order to perform experiments that yield quality results, samples need to be stored properly in order to preserve their activity or integrity. Beginning students and…
You’ve spent days, perhaps weeks or months squirrelling away tubes of preserved tissue in the dark drawers under your laboratory bench like the trophies of a demented serial-killer. Hours have been spent in histology in the processing, embedding and sectioning onto slides. Finally, like a warrior victorious in battle, you hold aloft your thin glass…
Here, we share a protocol for a midiprep, which, if not faster, gives a larger plasmid DNA yield than any commercial midiprep kit.
Summertime… The birds are singing, the trees are growing. Your tissue culture has sprouted yeast contamination, your yeast culture is happily growing bacteria. Your bacterial culture was growing calmly and predictably, dividing every twenty minutes, but suddenly its optical density has dropped, and it’s full of some sort of filaments and clumps. Or you did…
Before using any Biological Safety Cabinet (BSC) for the first time, have a person with working knowledge of the machine give you an overview of how to use the cabinet. Different labs have different protocols in regards to running the cabinet, disinfecting the cabinet, determining which pathogens that may be used in the cabinet and…
The last step in western blotting is imaging the blot – this is the moment of truth, when you finally get to see the results of the experiment you’ve been working on for so long! There are a variety of different ways to image your blot. The method you choose will largely depend on the…
As we learned in my previous article, cell culture hoods have many names. As if that wasn’t enough, they are all-too-often misunderstood and mistreated, which can lead to dangerous situations harmful for both the worker and the general lab environment. Here are three common ways that workers abuse biological safety cabinets; make sure you don’t…
Biological safety cabinets, laminar flow hoods, clean hoods and culture hoods are all common names for those essential pieces of equipment that you use in cell culturing. The terms are used inter-changeably, but in fact there are lots of different types of culture hoods, each of which does a different job. Knowing which is which…
The first step in most Western blotting experiments is lysing your cells to extract protein. You need to break open your cells in order to be able to isolate the proteins, and you need to do this with the least degradation and the most reproducibility possible. Depending on what your starting material is, there are…
There are several yeast transformation protocols around, and most of them require a lot of steps: overnight starter culture, dilution and growth to logarithmic phase, several washes, and so on… These protocols work very well since they have been optimised for maximum transformation efficiency, which is needed for applications like library construction. But they are…
Proteases: wild, mysterious, destructive. What are these untamed elements ravaging your precious lysate? How can a drop of EDTA or a smidge of “cocktail” protect that sample, which is gently cradling your hopes, your dreams, and your desire to survive the next lab meeting? Brace yourself for a biochem flashback: in this article, we’ll explain…
After having discussed what epigenetic mechanisms are and how we’ve learnt about what they do, it is now time to look into how epigenetics affect our lives if things do not go the way they are supposed to go. I hope I have convinced you that epigenetic processes are vital for an organism, in development…
Epigenetics is the most rapidly expanding field in biology. In the second article in this series, I discussed which experimental techniques have been crucial in gaining insight into epigenetic processes. I will now shed light on what those and other methods have taught us. As described in the first article, it has been long understood…
In the past decade, important advances have been made in the field of epigenetics. Obviously, unraveling epigenetic mechanisms has been greatly facilitated by technological developments. I’ll try to give you an impression of the types of experiments that have helped fuel those new and exciting insights. Yevgeniy Grigoryev has recently written an article on DNA…
These days, epigenetics is a fast moving field. I don’t remember having learnt about it during my biomedical studies, some 10 years ago. Nowadays, there seems to be no way around it when studying health and disease. Increasing interest combined with recent technological breakthroughs have led to quickly expanding knowledge of its abundant and important…
A young laboratory technician was puzzled by his plates when he pulled them out of the warm room. They never looked that cloudy and fuzzy before. He brought them to his lab director, who shook her head sadly; together, they threw the plates away. Does this story sound familiar? What probably happened was an all-too…
Mycoplasma infections are very, very bad news; these special prokaryotes can rapidly spread through your cell culture and inhibit cell proliferation, induce apoptosis, cytokines and radicals, and otherwise transform your cells. Worst of all, since contamination is not easy to spot, you may not realize your culture is contaminated until it’s too late. The 100…
Commercial kits for isolation of large quantities of plasmid DNA generally rely on standard alkaline lysis followed by an affinity chromatography column-based method to purify DNA. Compared to traditional cesium chloride banding or PEG precipitation of plasmid DNA they are a breeze, and have the added benefit of avoiding use of toxic or hazardous chemicals…
Western blots may be great for visualizing protein expression, but they can be a perfect way to waste your precious antibody stocks if you follow the normal protocol. Thankfully, you don’t have to follow the normal protocol any more; here’s how to get great blots with a fraction of the antibody usage. I have tried…
You’ve masterfully run and transferred your gel, and now it’s time to probe and quantify your protein(s). You’ve got your antibodies and ECL ready to go. Substrate – check. Film cartridge – check. Darkroom – ah yes, that magical place where night vision goggles are required to navigate a veritable minefield of potential chaos. It…
Epigenetics is the study of heritable changes in the phenotype of a cell or an organism that are not encoded by the genome (hence epi which means ‘above’ in Greek, and genetikos which means ‘origin’). In this article, we’ll discuss DNA methylation, a common epigenetic modification: what it is, how to detect it, and how…
When running a quantitative Western blot, it’s crucial that your sample preparation is consistent. Incomplete protein extraction from one sample will skew your results when you compare it to the protein content of a sample that was extracted more thoroughly. And after the protein extraction, it’s important to handle the samples in an identical manner…
Working with RNA? What fun! Those little, nearly indestructible RNases are everywhere – on your skin and mucous membranes, in the water and (some of the) enzymes you use, on lab surfaces, even in airborne microbes! Here are 10 ways to keep the RNases at bay, and keep your precious samples safe:
When it comes to choosing a molecular weight marker to run on your SDS-PGE gels, there are a lot of options out there. How do you know which one is right for you? Read on for tips on what to consider when choosing a standard for your protein gels. Before you go about selecting a…
So, you’ve done your experiment, prepped your samples, and run your SDS-PAGE gel. Now it’s time for the all-important transfer step, that tricky point that will determine the quality of your Western blot. Transfer times are empirical and based on your own particular samples, which means that there is no easy way to determine how…
I think that transferring Western blots is one the most enjoyable tasks to do in a lab: it’s quick, it’s messy, and on some gleeful level, it feels like a child’s art project gone wrong. Of course, it’s also finicky and slippery and prone to tiny pitfalls that can noticeably affect the quality of your…
For Western blot data to be reliable, it is important that you load known amounts of sample into each lane of the gel. This is of particular importance if you are doing a quantitative blot, where you really need to be able to compare band intensity in each sample. In this article, we’ll talk about…
While Luria-Bertani broth (LB) has long been the fuel that powered Molecular Biology and Biochemistry, there is an increasing movement towards more specialized and complex bacterial media formulations such as Terrific Broth (TB), Plasmid DNA Media (PDMR), and Autoinduction Media (ZYP-5052). These media formulations optimize E. coli cell growth and performance utilizing specialized carbon sources…
I recently introduced you to the concept of polarising microscopy. Naturally, if evaluating refractile material is an everyday part of your research, it is definitely worth investing in a professional polariser modification for your microscope. But if you only use a polariser occasionally, this might not be the best use of your lab’s money. In…
Tandem affinity purification is a development of existing techniques for purifying protein complexes from cells in physiological conditions. It was first described over ten years ago and has become a commonplace laboratory tool. In this brief article I’ll introduce the basic technique and describe some of its advantages. Biology is a team game. Most biological…
When you stop to think about it, tissue slices for immunohistochemistry (IHC) undergo quite a lot of handling. From chemical reactions to washes – even manipulations and transfers between baskets and microtubes – final analysis is often hours away from the initial step of taking a tissue slice. Properly fixed tissue has to be robust…
Polarising microscopy involves the use of polarised light to investigate the optical properties of various specimens. Although originally used predominantly in the field of geology, it has recently become more widely used in medical and biological research fields too. Polarising light microscopy is a contrast-enhancing technique to allow you to evaluate the composition and three-dimensional…
There’s more to mammalian cell culture than just making sure that your cells don’t die. It is a lot like taking care of children. You have to feed them, make sure that they’re growing well, and keep them under constant supervision. If the cells are put through extreme conditions (over-confluency, media-deprivation, inaccurate temps, etc.), their…
Do you use human cell lines in your research? Well, keep reading because this may be the most important article you will ever read in your research career. It is estimated that 18-36% of all actively growing cell line cultures are misidentified and/or cross-contaminated with another cell line (1). For researchers, this could mean that…
Microarrays are one of the most in-depth ways of determining cellular gene expression levels of thousands of genes simultaneously. They are able to help determine: Gene function and cellular processes Gene regulation and interactions Gene expression levels in different cell types and how this expression is altered by the addition of various compounds or disease…
For applications such as site-directed mutagenesis, it is often recommended that you use a proofreading polymerase (also known as high-fidelity polymerases) to minimize the risk of introducing unintended point mutations. But what is a proofreading polymerase? What makes them different from other polymerases? And when should you use them? Read on to learn more… What…
Most site-directed mutagenesis protocols strongly recommend that you use only PAGE- or HPLC-purified primers to mutate plasmid templates. Using purified primers is supposed to minimize the introduction of unintended mutations, thus drastically improving the probability of generating your desired mutant. However, specially purified primers can be extremely expensive, and take longer to synthesize than standard…
RNA interference (RNAi) may have originated as a defense mechanism to protect cells against foreign genes introduced by viruses. This concept has since been put to use to create a powerful experimental tool for investigating gene function in organisms. Small-interfering RNA (siRNA) libraries for investigating genome-wide function can be produced by chemical synthesis of probes…
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