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Plants are incredibly organisms. Not only do they provide atmospheric oxygen, but, in the case of legumes, they can transform atmospheric nitrogen gas to ammonia, which can then be consumed by humans. How does this happen, you ask? It’s all thanks to bacteria and the process of nodulation. Deep Breaths Nitrogen is an incredibly important…
Restriction digests are the cornerstone of many techniques. Here are some great tips on setting up and troubleshooting them.
Organoids are a developing star of research. They can be grown to represent the majority of mammalian organs and have a wide range of possible applications. More realistic than simple monolayer cell culture, they offer an in-between step that reduces the need for animal models and simplifies (although doesn’t completely remove) ethics paperwork. They are…
After a late night transformation you realise you have forgotten to make any plates. Should you use the old stash of amp plates you found in the back of the cold room?
Wound healing assay is one of the earliest and simplest in-vitro assays for studying cell migration. Discover if this assay is right for you and learn how to set it up to get reproducible data.
Resistance to the antibiotic ampicillin is commonly used as a selection marker for plasmids in gene cloning and protein expression in E.coli and other bacteria. While it can be incredibly useful tool, there can be problems using this selection marker that you need to be aware of if if you plan on using it. This…
A Spot of History Most of the biomedical methods used started as a curiosity. Then the one-off gains a limited use, the technology then progresses until its use becomes widespread. Just think about the arch from the curious polished glass spheres, used by Antony Levnhook to look at animalcules, to modern microscopes. The same story…
Antibiotics are used in a wide range of techniques in molecular biology including molecular cloning and are important for treating pesky mycoplasma contamination in cell cultures. They can also be used to maximize your plasmid yields by reducing protein synthesis, in certain circumstances. The aim of this post is to provide an easy reference to…
The Phobia of RNases My first experience of troubleshooting in vitro transcription came when I was synthesizing RNA In-Situ Probes for the first time. A lab mate ominously warned me that I had just returned to lab after a bout of flu and that meant I’m a walking talking factory of RNases. I went ahead…
Purifying a new protein is no easy feat. Finding combinations of protein purification buffer, salt, detergent, and stabilizing agent to get high yields of squeaky-clean protein can become tedious. Few things are as bothersome during this process as Heat Shock Protein (HSP) contamination. But worry not, we’ve got some handy tips to avoid HSP contamination…
Want to do some epigenome editing? Discover the usefulness of catalytically inactive (dead) Cas9.
The advent of Next Gen Sequencing (NGS) has been truly amazing. One of the marvels that is often overlooked is how advances in DNA extraction technology have helped streamline NGS workflows. The original standard – phenol/chloroform extraction – is not well suited to the automated nature of today’s sequencing workflows (though with the emergence of…
Overnight ligations are inconvenient — especially when they fail. Luckily, there’s a straightforward way to faster DNA ligations. This article highlights the secret ingredient to faster ligation reactions and offers some tips and caveats on its use. For a general overview of DNA ligations, see here and here. Buy a Quick Ligation Kit The most…
One of the most widespread protein laboratory accessories are the MWCO (molecular weight cut-off) centrifugal filters which are commonly used for concentrating protein, as well as DNA. They are available commercially with different cut-offs including 3kDa, 30kDa, 50kDa, 100kDa, and so on. These little devices are expensive and hence demand proper usage and care to…
What Is Air-Liquid Interface Culture? Long gone are the days where scientists had to rely on 2D cultures of immortalized cell lines to learn principles of human biology. Today, we have a variety of cell culture systems that come closer than ever before to mimicking the structure and function of our body’s organs. One example…
What Do We Mean by Diagnostic Sensitivity? In clinical diagnostics, questions about the sensitivity of an assay will inevitably surface. But what does “sensitivity” mean exactly? The lowest quantity of the given analyte that an assay can detect is often called sensitivity – and to be clear, this quantity is the analytical sensitivity or Limit…
Cell Biology is entering the Age of Light with a spectrum of new optogenetics tools available to control protein function using light. Once the remit of neuroscientists [1], the past decade has yielded a bounty of novel light-controllable domains that are now being leveraged to illuminate the dark corners of basic cell biology [2,3]. The…
In research, choosing a model system is like choosing a partner – you want it to be a perfect fit. If you are attempting to solve problems such as finding unknown proteins in known processes, investigating unknown functions of known proteins or correlating cell biology to a function for which you want a relatively simple…
Let’s face it: the nature of behavior itself is inherently variable, whether it’s the heterogeneous socializing behavior of humans at parties, the complex aggressive behavior of rodents when they perceive a threat, or the intricate courtship behavior of insects during their mating dances. Because of this variability, the struggles associated with trying to (successfully) reproduce…
How to Prevent False Results in Colony PCR Colony PCR saves time and reduces costs by eliminating the need for plasmid purification. However, confounding results abound — but only if you fail to anticipate them. This article outlines the major perpetrators of false results and how to prevent them. For a more general overview of…
What do DNA mini preps and protein immunoprecipitation experiments have in common? They start differently, but they end with the same, critical stage – elution. But what exactly is elution, and what is the point? The Terminology First, let’s start with some basic terminology: Elution – extracting one material from another by washing with a…
What is Isothermal Titration Calorimetry? Isothermal titration calorimetry (ITC) measures the heat generated (or absorbed) when one solution is titrated into another. Most commonly, a small molecule or peptide is titrated into a protein. If the molecule binds to the protein, heat is given off (or absorbed) with each injection, until the protein is saturated.1…
Nanopore is a relatively new sequencing platform and researchers are still trying to optimize the protocol for their own specific applications. In our lab, we work primarily with metagenomic samples and use the 1D sequencing kits. Over the past year, we have optimized this technique. To check the quality of the Nanopore library preparation we…
In the first article of this series, I introduced microscopic parasites that infect greenhouse plants. Initially, my goal for Part 2 was to list all the main types of infestations, but I quickly realized to do so would result in a textbook. Thus, this article will be about the most familiar and easy to identify…
If you look at the composition of peripheral blood, using hematology microscopy, you’ll see that it’s composed of multiple different cell types, including monocytes. It’s possible to isolate these different components to study and experiment on them directly. So, if you’ve done a few experiments and had fun with THP-1 cells, you can move on…
CRISPRa allows you to activate or overexpress genes in a more endogenous manner. Find out the steps to getting started.
As the vast majority of bacteria cannot be readily cultured in the laboratory [1], culture-dependent methods to investigate bacteria grossly underestimate the diversity of bacterial communities. To investigate unculturable bacteria without isolating them, culture-independent methods such as sequencing have been used. Unculturable bacteria can be identified by PCR amplification and sequencing of housekeeping genes such…
Cloning Strategies – a Whole Lot of Options to Choose Molecular cloning has come a long way from simple restriction digestion-ligation cloning strategies to a large number of highly efficient alternatives. Broadly classified, cloning techniques can be divided as sequence dependent and sequence independent strategies. Sequence-dependent strategies are based on restriction digestion-ligation techniques or site-specific…
In both the lab and field, it is important to know what species we are working with. While morphological data has always been a tried and true method of identifying species, DNA barcoding allows us to identify species when we don’t have that option (e.g. if we don’t have enough of a specimen to identify…
Level-up your troubleshooting ability by determining the success of failure of each stage of your CRISPR experiment.
What is the first image which comes to mind when you think about microalgae? Green scum that covers the surfaces of ponds? Unsightly stains on pavements and walls? Far from being a nuisance in ponds, lakes, drains and on surfaces, microalgae are fascinating microorganisms which are used to understand various biological processes. Microalgae have been…
Studying nucleic acid interactions with proteins can be accomplished using a rapid and efficient electrophoretic mobility shift assay (EMSA). This method is essentially an agarose gel electrophoresis technique that detects protein:nucleic acid interactions, as the mobility of the labeled nucleic acid will be retarded if bound to a protein (compared to unbound DNA). A lesser-known…
Reduced-representation genome sequencing has been one of the most important advances in the last several years for enabling massively parallel genotyping of organisms for which there is no reference-grade genome assembly. An implementation of the approach known as ddRAD-seq, first conceived in the Hoekstra lab at Harvard, has been widely adopted by the plant and…
Out with the Old… Well-based assays have been the standard for common laboratory experiments, such as fluorescence cytometry. A researcher places a small amount of sample into a well on a plate and assays it, which produces a single data point. However, this so-called single data point is actually an average of the measurements of…
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…
In theory, the greenhouse is a controlled laboratory environment where only the organisms you’ve introduced live. But in practice, just as other laboratory environments suffer from ‘unwelcomed guests’ (e.g. contamination and infestation), greenhouses are not always as sterile as you would like. To avoid any experimental issues, you have to be vigilant about these pesky…
WGS technologies have seen significant progress since the completion of the Human Genome Project in 2003. First-generation Sanger Sequencers were limited by lengthy run times, high expenses, and throughputs that read only tens of kilobases per run. The arrival of second-generation sequencers in the mid-2000s brought about the plummeting of sequencing costs and run times,…
In whole genome sequencing (WGS) initiatives it is not enough to simply sequence the whole length of the genomic DNA sample just once. This is because genomes are usually very large. The human genome, for example, contains approximately 3 billion base pairs. Although sequencing accuracy for individual bases is very high, when you consider large…
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…
Generally speaking, fruit flies are a great model system. Not only are they small, thus taking up very little space in the lab, but their adult lifespan is only 40-60 days, so you can track age-dependent changes without having to wait months and months. Fruit flies also display complex behaviors and more than 75% of…
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