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Fluorescence is one of the most important and useful tools in a biologist’s toolbox. In biology, nearly every field, from physiology to immunology, uses fluorescent molecules (aka fluorophores) to detect proteins. However, the specific science behind how fluorescence works can be confusing or overlooked. Have no fear! In this article, we break down key points of…
You have probably run a standard agarose gel hundreds of times. They are great for visualizing small DNA fragments up to 10 kb, but what if you want to examine really large pieces of DNA or even whole chromosomes? This is where pulsed field gel electrophoresis (PFGE) comes in! While the equipment required to run…
In the 30 odd years since its invention, the polymerase chain reaction (PCR) has become the bread and butter technique of molecular biologists. The secret to its indispensability lies in its simplicity and versatility. Numerous variants of the technique have been developed; one of these, real-time PCR, has become the method of choice for quantitative…
In this article I will not talk about ‘wild’ proteases, which destroy cellular proteins in your lysates like wolves destroy sheep. Instead, I’ll be talking about the shepherd dog proteases—purified, tame and useful to digest proteins your research. In Protein Research and Crystallization Several programs can predict your protein domains. However, we wet biologists know…
If there is one profession that benefited from globalization, it is the medical writer. While the university research groups shrink and global biomedical companies fire their research stuff, medical writing companies are expanding, providing stable jobs with good salaries. The American Medical Writers Association (AMWA) reported in 2011 that the median salary of an experienced…
Has your supervisor asked you to do a polysome profile? Are you attempting them but running in to problems? This article will help explain what they are, the basics of the protocol and provide some helpful tips (including some things that I wish I had known when I first started out). Polysome Profiling: The Basics…
This is the first in a three-part series on the transformation of E.coli. By the end of this, you should be an expert on E.coli transformation and on which strains to choose for different applications. If you’re already an expert, I hope it’ll be an enjoyable refresher for you. In either case, please comment below…
Mass Cytometry is a relatively new technology which has recently featured in many high-impact journals. You may have read about instruments including the CyTOF, CyTOF2, and more recently, the Helios. With these instruments becoming more widespread, you might find yourself asking, what is mass cytometry, and what can it do for you? The Basis: Conventional…
To isolate your protein, you are going to need to tag it with something that will allow you to fish it out of the bacterial protein soup. This is where transition metals can help you out!
The Gram stain is another commonly used special stain in the histology lab. Why use a Gram stain? The Gram stain is a type of differential staining technique which represents an important initial step in the characterization and classification of bacteria using a light microscope. It is named after a Danish scientist, Hans Christian Gram,…
While overexpressing a gene of interest can provide a look into its role in a cell, sometimes it is necessary to control the expression of a gene. You may want to dictate the timing of the protein’s expression or lower its expression level to adequately understand its function. This is particularly relevant when studying genes that…
Interested in whether your protein uses oxygen to mediate reactions? Wondering if oxygen is keeping your enzyme from its duty? Then what you need as an anaerobic tent! These tips provide some basic knowledge to help you perform experiments using an anaerobic tent. What is an anaerobic tent? Most biologists who work in oxygen-free environments…
Get some ideas on what CRISPR can do for you and what using it involves.
Are you an assiduous biologist who prefers label-free imaging methods for biological samples analysis? Raman spectroscopy offers you a wonderland of imaging technique with unlimited benefits. To start with, Raman Spectroscopy is a spectroscopic technique based on inelastic scattering of monochromatic light usually from a laser in the visible or near infra-red part of electromagnetic…
I’ve noticed that there exists some ambiguity about the different terms relating to homology. I’ll try to break them down here, with significant help from Genome Biology.
When you first start out using a microscope, you might only adjust the eye pieces, objectives, and the focus controls. However, you shouldn’t overlook the microscope condensers as they are an important part of the whole optical system of a microscope.
In Part 1 of this article, I introduced you to using code for basic image manipulation in ImageJ and working with the command recorder to expand your coding vocabulary. I covered how to make a simple macro, how to edit it and then save it to be run again another time. If you skipped the…
In part I, I answered the question, “How do proteins in thermophiles survive under high temperatures?“ In this part, I’ll look look at how nucleic acids survive -thrive, even- in conditions that are too hot for most of us, but ideal for a number of organisms, including the one that gave us Taq polymerase and…
Before I get into today’s topic, please allow me to digress a bit and start with a few sentences that sum up the polymerase chain reaction (PCR); the grand-daddy of molecular biology. PCR, a method that is at the heart of modern day molecular biology discoveries, is a process that amplifies genetic material through our…
The flow cytometer that we have all grown to know and love may have only come into its own in the 1990’s, but who would have known that the first cell sorter was invented as early as the 1950’s? With the recent death of one of the key developers of fluorescence activated cell sorting (FACS),…
Every protocol for single cell PCR can be broken down into two steps. In the first step, the cells are isolated by micromanipulation, laser capture microdissection, flow cytometry, or by direct micropipetting. Next, the genetic material is processed by PCR to amplify your sequence of interest. Here, we’ll go through the different options for isolating…
Want a PCR method that gives you high sensitivity and is cost-effective? Then you might want to try Mediator Probe PCR. This method gives you sensitivity and limit of detection comparable to that of dual-labeled hydrolysis probes without the high cost. We all know how expensive small batch probe synthesis orders can be, especially when…
As researchers, we are constantly on the lookout for new and improved ways to analyze, detect and quantify our favorite protein or gene. Luckily, we don’t always need to reinvent the wheel! PCR-ELISA is a good example of where two commonly used techniques have been merged together to create a very powerful analytical tool. What…
The completion of the Human Genome Project in 2003 ushered in a new era of rapid, affordable, and accurate genome analysis—called Next Generation Sequencing (NGS). NGS builds upon “first generation sequencing” technologies to yield accurate and cost-effective sequencing results. Fred Sanger sequenced the first whole DNA genome, the virus phage ?X174, in 1977. In that…
The wide range of applications of PCR has led to an ever-growing list of variants of the technique. While some are optimizations to suit specific requirements and are very similar to basic PCR, others completely turn the technique on its head to formulate novel creative applications in various fields. This article lists some variants of…
PCR (Polymerase Chain Reaction) is a biochemical technique developed by Kary Mullis in 1983 that is used to create large quantities of a sequence of DNA. Since this method of mass-producing DNA was first introduced, it has become significantly less labour intensive, more economical, and more routine. The technique relies on a few key players…
The Scene of the Crime The body of a woman is found behind an abandoned warehouse on the outskirts of town. Forensic experts carry out a technical examination of the scene and suggest strangulation as the cause of death. The absence of bloody footprints or weapons means there are no obvious leads to the killer….
A wide variety of enzymes are available for PCR and RT-PCR and the optimal choice depends on a range of factors specific to your experiment. Some of these factors will now be explored to help you to make the most suitable and cost effective choices when ordering. PCR Type and Other Factors to Consider First,…
So you’re designing a new experiment that requires PCR quantification. You used to have only one method to choose from, but now you have two – Quantitative Real-Time PCR (qPCR) and Digital PCR (dPCR). Which one is right for your application? Both methods have good quantification, sensitivity and specificity for most applications. They are compatible…
Every PCR battle is the same: Too little amplification of your target DNA versus too much amplification of off-target DNA. But you can win the PCR battle and amaze your co-workers by mastering the use of PCR additives. PCR additives usually work one of two ways: 1) By reducing secondary DNA structures and thus increasing…
Bisulfite pyrosequencing is becoming a routine technique in molecular biology labs as a method to precisely measure DNA methylation levels right down to the single base. The technique allows for detailed and high resolution analysis of DNA methylation at specific genomic regions. How to detect the 5th base? Methylation of any of the four nucleotides…
To answer some of the more interesting research questions, you often need to get a good look at what’s going on inside the cell. Whether you’re running a Western blot or measuring enzyme activity, many assays require access to the materials (e.g. proteins, DNA, subcellular fragments) contained within the cell walls. There are several ways…
Isolating pure DNA is key to many downstream applications for molecular biologists. Isolating large quantities of pure DNA used to be a laborious task. But thanks to commercially available kits, older methods have been streamlined to allow efficient recovery of pure DNA. In this article, I will talk about a method called DNA gel extraction,…
In her article How to Get Perfect Protein Transfer in Western Blotting, Emily Crow recommends Coomassie staining your gel after transfer to the membrane to check the quality of the transfer. A good transfer should not leave behind proteins and PVDF membrane, stained by 0.1% Ponceau S in 5% phosphoric acid and destained with water…
2D gel electrophoresis (2DE) is a key technique for purifying individual proteins from complex samples based on their isoelectric points and molecular weights. Simple enough in theory, but as the plethora of protocols and articles shows, 2DE demands patience and meticulous optimization. But whether your samples are human sera or HUVEC lysates, 2DE uses these…
If you have ever worked with RNA, you know about DEPC (diethylpyrocarbonate). You add it to water at a concentration of 0.1%, shake or stir, incubate at 37°C for two hours or at room temperature overnight and, as if targeted by a magic bullet, the RNAses that may have been in the water are gone….
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…
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…
One of my colleagues, a very good molecular biologist, told me that the only time she uses chemistry is when she needs to calculate molarities. I, of course, scoffed at this statement, and tried to remind her of all the chemistry she uses daily. True, I may be a bit biased since I am a…
Diving deeper into protein kinases, explore the vast realm of known kinases, their amino acid targets, and the intricate process of target selection.
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