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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…
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,…
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…
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. …
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…
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…
Why confocal? The standard fluorescence widefield microscope described in our last article has one major disadvantage: it collects not only the desired image information from the focal plane, but it also records a large amount of out-of-focus light, leading to a blurred image. In the 1950’s, system developers started thinking about how to get rid…
The “sequencing-by-synthesis” technology now used by Illumina was originally developed by Shankar Balasubramanian and David Klenerman at the University of Cambridge. They founded the company Solexa in 1998 to commercialize their sequencing method. Illumina went on to purchase Solexa in 2007 and has built upon, and rapidly improved the original technology. Millions of reactions and…
Arguably, molecular biology is impossible without microbiology—even if you work exclusively with transgenic mice, you may one day need to amplify a vector in E. coli. And microbiology is definitely impossible without good aseptic technique. The main principle of good microbiological practice is a zero tolerance approach: it’s good to be a little paranoid about…
Think before you start! Before you even start preparing your samples, you should think about the choice of microscope for image acquisition. Manufacturers offer an ever increasing range of light microscopes- some of which may already be available in your research institutes. To help you get the best out of your imaging experiment by making the…
Don’t be left in the dark! Navigating through the sea of parameters that can aid or abet successful microscopy can perhaps seem daunting. It may be tempting to assume you can just sit down at a microscope, look at your sample, and obtain beautiful images simply by hoping for the best. However, even a basic…
Much more than just an archive, The Cell: An Image Library-CCDB (Cell Centered Database; ‘The Cell’) serves many additional purposes. Whilst many researchers use The Cell to organize their own images for research and archival purposes- the real value of The Cell is the ability to share those images with other researchers. In many scientific…
Protein tags are invaluable tools for the modern biologist, particularly if you work on one of the 99% of proteins for which there isn’t a nice antibody readily available. If you want to purify large amounts of your protein of interest, detect it by western blot or fluorescence microscopy, or identify its potential binding partners,…
Next Generation DNA Sequencing (NGS) is a revolutionary new technology that provides biologists and medical scientists with the ability to collect massive amounts of DNA sequence data both rapidly and cheaply. This technology is having a huge impact on many aspects of biology and medicine because it can be applied in so many different ways….
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