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What’s The Problem With Ampicillin Selection?

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…

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Should You Switch from Wet to Dried Blood Samples?

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…

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How to Preserve Microorganisms: the long (and short) game

Given the importance of microorganisms as cell factories in biotechnological applications and as model organisms for the study of various biological processes, the preservation of microorganisms plays a key role in ensuring reproducible results and continuity in research. Maintaining a library of microbial stocks also enables microorganisms to be easily stored and retrieved, as compared…

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A Beginner’s Guide to How Blunt-End Cloning Works

Blunt and sticky might sound dull and dirty but knowing how these different cloning methods work is important when choosing which method to use. Here we give you a guide to how blunt-end cloning works. Blunt-end cloning is the cloning of DNA fragments containing no unpaired bases at the 5’ and 3’ prime ends (i.e.…

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Antibiotics Used in Molecular Biology

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 yeilds by reducing protein synthesis, in certain circumstances. The aim with this post is to provide an easy reference to…

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Top Tips for Troubleshooting In Vitro Transcription 

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…

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Dead Useful: CRISPR-Cas9 Epigenome Editing

Given the rapid pace with which genome editing technologies have been developed and adopted, it comes as no surprise that the original CRISPR-Cas9 system has been successfully modified by some very clever scientists. No longer are we limited to the ‘simple’ case of editing the genetic sequence of your biological system of choice, but there…

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DNA Extraction for Next Gen Sequencing

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…

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Faster Ligations: PEGing down the Secret

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…

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Spinning Around: Tips and Tricks for Using Centrifugal Filters

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…

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Staying Alive: Tips for Air-Liquid Interface Cultures

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…

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How to Totally Nail Your First in situ Hybridization

It’s Monday morning and you’re about to start your first in situ hybridization (ISH). You glanced at the protocol on Friday and it seemed pretty easy. You just do a lot of washes and incubations, right? WRONG. ISH can be a nightmare. If you’re not well prepared, you’ll most likely be tearing your hair out…

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Laemmli Buffer: What Is It for Anyway?

Electrophoresis encompasses a wide range of techniques in which charged biomolecules in a liquid, a solid, or a semisolid solution can be separated by size under the application of an electric field. The most common application of electrophoresis for the separation of proteins is SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), which has been previously covered here.  …

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Getting Sensitive: Diagnostic Sensitivity and Specificity Simplified

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…

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Designing a LOV2 Domain-Based Toolkit

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…

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C. elegans: The Elegant Model System

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…

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Five Factors Affecting Your Mouse Behavioral Studies 

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…

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Controls for Immunofluorescence: A Beginner’s Guide 

Immunofluorescence staining is a popular and extremely powerful detection method. However, achieving publication quality immunofluorescence or fluorescent antibody staining can get tricky. It’s therefore important to ensure you have the right controls for immunofluorescence.   Since immunofluorescence staining is a long process with many steps, it is always advisable to spend some time optimizing each…

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How to Prevent False Results in Colony PCR

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…

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