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It takes some time to complete any professional education beyond a bachelors degree. So what to do if you’re not absolutely sure about spending the roughly sixty-or-so-months it takes to achieve a PhD? A hands-on internship or lab rotation is an excellent way to investigate science as a career. In a few short months an…
Every once in a while a big case of scientific fraud reaches public attention. Does that mean these well-known cases are exceptions, a few rotten apples…or might the rest of the fruit bowl also be affected? A major part of a scientist’s work is to secure funding for future research. Obtaining funding is strongly connected…
The stability of an antibiotic depends on its chemical structure, method of isolation (from natural sources or chemical synthesis), and the mechanisms of inactivation. First generation antibiotics isolated from natural sources, such as penicillin, are the most unstable, followed by its semisynthetic derivatives (such as ampicillin and carboxycylin). Aminoglycosides (kanamicin, spectinomycin, etc.) are more stable….
We’ve all been there. Digging through the -80°C freezer, fingers about to get frostbite as you scrape the ice off a tube to read an illegible plasmid name, hoping it’s the right plasmid with little or no knowledge of how it was cloned in the first place. And this is the starting material for your…
RNA sequencing (‘RNA-seq’) has become one of the most widely used applications for Next-Generation Sequencing. RNA-seq can provide gene expression data more cheaply than microarray, at greater sensitivity, and without the biases inherent in an assay based on quantifying nucleic acid hybridization. RNA-seq can also provide data about alternative splicing, allele-specific expression, expression of non-annotated…
In Part 1 of this Guide, we learned about the importance of support, resources and objectives when choosing a fluorescence microscope. We continue this guide by looking at everything from filters to warranties. You should get these filters… Quality of the barrier filters and dichroic mirrors are the deciding factor on how well you can…
You’re a senior postgraduate student, a post doc or a junior PI with little knowledge on microscopes and someone between Senior PI /Dean level approaches you with this: “We have now the funds to buy the fluorescence microscope someone once told me we need. Can you handle this please? Oh, by the way, the funds…
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…
Transfection of animal cells has proven an invaluable tool in studies of gene expression, cell behavior, cell processes and molecular genetics. Essentially, transient pores are opened within the cell’s lipid bilayer, allowing insertion of nucleic acids (DNA, RNA, siRNA, RNAi), proteins, nanoparticles, and even antibodies into the cellular milieu. Transfection can be achieved using many…
Thirty-five years ago, Dr. Janet Davison Rowley sat at a microscope in her lab at the University of Chicago and made a remarkable discovery in cancer biology: that leukemia is caused by the translocation of a chromosome. In other words, it is a disease of the DNA. Today, thanks to next generation sequencing (NGS), we can zoom in…
Tandem affinity purification (TAP) is a versatile technique allowing the isolation of proteins for various purposes including Western blot and mass-spectrometry. The target protein is fused with protein A from streptococcus and the calmodulin binding domain, which together comprise the TAP-tag (for an introduction to TAP-tagging, see this article). To purify a TAP-tagged protein from…
Coating (or ‘subbing’) slides for immunohistochemistry can be the difference between having an organized set of tissue slices ready for microscopy- or watching them detach and float away during a wash. It takes a lot of time to place tissue slices in correct anatomical order, aligned right-side up and flat. To the naked eye, all…
Having to make stock solutions is a part of everyday lab life…a tedious, but necessary, one. So why not make the process as streamlined as possible? Here are a few little tricks I picked up while I was still in the lab: What are your tips for making stock solutions?
Plasmids—the loops of DNA in bacteria that form the original foundation of biotechnology—were being discovered constantly in the 1940s and 1950s. The only problem was, they were called everything but. Series of scientists found bacteriophages and other strange loops of somatic DNA, and gave them a series of names, including: pangenes, bioblasts, plasmagenes, plastogenes, choncriogenes,…
To become an expert in managing people and projects together, a scientist needs a variety of important skills to succeed. One way to add to an already impressive skill set is by mentoring others through internships. Students take internships to be exposed to new things. Mentors give internships to inspire others to do research. They…
After years of hard work in the lab, having to sit down and write everything up can be a daunting and difficult task. Here are a few tips that I picked up during my thesis write-up that might help to make the process that little bit easier. Back up This is probably the advice most…
Visualization of DNA in gels is one of the most common procedures a molecular biologist can perform. In a good day, I can run at least 4 different DNA gels! When I was trained, ethidium bromide (EtBr) was the only viable option to easily visualize small amounts of electrophoresed DNA. However, safer and more sensitive…
So, you’ve just received a call from the core facility that you hired to prepare and sequence your libraries. The facility director tells you that the sequence data from your next generation sequencing (NGS) experiment does not look good. You panic and, perhaps, let loose a scream of frustration—aaarrrrggghhhh! This project was going to be…
Ever wake up especially groggy in the morning, finding it takes a few minutes and a few eye rubs to be able to decipher the numbers on your alarm clock? Our eyes have the ability to resolve an image, so that you can observe separate objects and details. Similarly, microscopes have a parameter of resolution:…
Whether you’re employing sequencing gels, Sanger-based methods, or the latest in pyrosequencing or ion torrent technologies, obtaining, manipulating and analyzing your sequences has never been easier. Depending on what your goals are, you need to understand the pros and cons of the software. There is a lot of software out there, so do you your…
In the same way that you should ‘Think Before You Fix’, the choice of embedding media should be dictated by your required end-point. The basic principle is that by processing tissue into an embedding medium you harden the tissue and provide support protecting it from the mechanical forces associated with sectioning. Parma ham and steak…
Fortunately, those of us who have learned how to sequence know that aligning sequences is a lot easier and less time consuming than creating them. Whether you’re employing sequencing gels, Sanger-based methods, or the latest in pyrosequencing or ion torrent technologies, obtaining, manipulating and analyzing your sequences has never been easier. We’re going to take…
LB medium is a staple in virtually every lab. It’s commonly used to propagate E. coli, and as such will be used frequently by any lab that does cloning. Chances are, LB broth or plates were one of the first things you learned to make as a newbie in the lab. Here are a few…
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….
Periodic acid-Schiff (PAS) is a commonly used special stain in the histology lab. Find out more about what this stain detects and how to use it.
The Human Genome Project was successful, but hard work. The major improvements to the technology were the increases in parallelization and automation. In 2003, just as the HGP completion papers were published in Nature and Science, ABI launched the‘3730XL’. It could run 24 96-well plates per day and generate around 2 MB of sequence. Some…
Keeping up with the latest developments in your field of research is important whether you are taking a sabbatical to travel the world or your funding has finally dried up. Whatever your situation, it is good to maintain a broad working knowledge of your field of interest and not leave yourself a mountain of reading…
Fluorescent-based microscopy techniques are some of the most common ways to visualize biological structures. Almost any protein- or nucleic acid-based molecule can be tagged with a fluorescent marker or dye and subsequently detected by a light microscope. This fluorescence is seen as a bright object against a black background, allowing for intense and clear images. …
Let’s face it: when you said you read that paper, what you really meant was that you read the abstract. And that conference you went to? You probably scanned the abstracts of the posters instead of actually attending the poster session and chatting with the presenters. It’s a dirty little secret and a time-saving tool…
It all started with proteins The earliest methods for sequencing were developed for proteins. In 1950, Pehr Edman published a paper demonstrating a label-cleavage method for protein sequencing which was later termed “Edman degradation”. Around the same time Fred Sanger was developing his own labelling and separation method which led to the sequencing of insulin….
Dichroic Mirror/Filter This is a semi-reflective filter which can also be referred to as ‘dichromatic beam splitter’. Unlike the Longpass filters which absorb light which is not transmitted (see Part 1 of the Glossary), these filters reflect light at lower wavelengths and transmit light at wavelengths above the ‘cut-on’ wavelength. As beam splitters, they are…
Co-immunoprecipitation is a method used to detect protein-protein interactions. While it can be wonderful when it works, there are many problems associated with this technique. One of the biggest problems that I have faced when using this method is contamination by the light and heavy chains of my precipitating antibody when performing western blots of…
Brightfield Illumination This defines the most basic method of optical microscopy using white light to illuminate the sample in the transmitted mode. Absorption and diffraction of the light by the molecules in the specimen generates the contrast in the image. Methods such as darkfield illumination, differential interference contrast and phase contrast help to increase the…
Collecting biological samples in the field can be difficult, since storage conditions outside of the lab are often less than optimal. Enter the Whatman FTA (Flinders Technology Associates) Cards. The Whatman FTA Card, a filter paper product manufactured by GE Health Care, is a paper matrix laced with a proprietary mixture of chemicals that lyse…
During my postgraduate studies, I did literally one PCR reaction with a pre-optimised protocol on a not especially difficult template. So my karma came back with vengeance, when as a part of my first postdoc I had to amplify a template containing a 35 bp-long GC-rich stem-loop, which proved to be extremely difficult. This was…
A revolution in 2005 The start of the NGS revolution was clearly marked in 2005 by the publication of the complete genome sequences of two bacterium (Mycoplasma genitalium and Streptococcus pneumonia) by 454 Life Sciences Corporation in one run of their Genome Sequencer with a 96% coverage at 99.96 % accuracy (Margulies et al. 2005)….
Have You Tried Trichrome? The trichrome stain is one of the most commonly used special stains in every histology lab. The pedantic meaning of the word trichrome is “three-coloured”, referring to how the technique differentially stains tissue samples in three colors. However, the term is now actually used to describe any staining method using two…
Every scientist, at one time or another, has avoided meetings with their PI. They are, however extremely important in shaping the direction and progress of your project. Having regular productive meetings with your PI will help maintain a positive working relationship and contribute to the success of your project. Keep the following in mind when…
Just before Life Technologies purchased the start-up company Ion Torrent, the fledgling company was dealing with a torrent of another kind—worldwide media interest in its new sequencing technology, which promised to bring the price of next-generation, massively parallel sequencing down to $1,000 per run. Since that dramatic announcement in the summer of 2011, Life Technologies…
Mammalian cell transfection is a technique commonly used to express exogenous DNA or RNA in a host cell line (for example, for generating RNAi probes). There are many different ways to transfect mammalian cells, depending on the cell line characteristics, desired effect, and downstream applications. In this article, I will review the different methods of…
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