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Search below to delve into the Bitesize Bio archive. Here, you’ll find over two decades of the best articles, live events, podcasts, and resources, created by real experts and passionate mentors, to help you improve as a bioscientist. Whether you’re looking to learn something new or dig deep into a topic, you’ll find trustworthy, human-crafted content that’s ready to inspire and guide you.
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…
The importance of epigenetics in biology is increasingly acknowledged (if you’re not convinced yet, read my crash course). One commonly studied epigenetic mark is CpG methylation: cytosines that are directly followed by a guanine nucleotide (indicated by CpG), can be methylated, unlike non-CpG Cs. Since attachment of a methyl group to a cytosine can affect…
Sanger sequencing is still a workhorse of most molecular biology labs. Even with the advent of next-generation sequencing we still need to sequence our clones and PCR products. In this article I have listed some of the tips and tricks we used in our Sanger services. (1)Dilution of BigDye: I’d expect this to be a…
The Basic Local Alignment Search Tool (BLAST) algorithm is at the heart of a free suite of online resources available through the National Center for Biotechnology Information (NCBI). While most researchers are aware of BLAST as a sequence alignment tool, NCBI’s BLAST suite offers so much more! I’ll cover in-depth how to use these resources…
The last two decades have seen a dramatic increase in the number of publications using immunohistochemistry (IHC) as a research tool to identify the spatial location of proteins of interest within cells, tissue sections and whole-mount preparations. Grinding and binding The advantages over ‘grind and bind’ methods are apparent, but the very best results will…
Finding a good primary antibody can often feel like playing Russian roulette. Nothing is more disappointing than buying a $300 antibody that doesn’t work for your use. There are some steps you can take, however, to increase your likelihood of success. And once you’ve found a good one, make it last by using the wet-chamber…
Calculations can be the bane of laboratory work. Fortunately, there are many easy methods to help you do the maths you need in the lab. Here, we tell you about the different ways to calculate primer concentration depending on the starting material. For all calculations, let’s assume we have 22 nmol of a DNA primer…
Alternative splicing events often occur in a spatiotemporal manner, and some are regulated by alternative splicing regulators, with striking variation across tissue types and developmental stages. Alternative splicing events are often differentially regulated across tissues and during development, as well as among individuals and populations, suggesting that individual isoforms may serve specific spatial or temporal…
Anyone who is involved in DNA sequencing in one form or fashion knows there are multiple ways to skin a cat: Sanger-based, next generation (NGS), and of course the new ion torrent sequencing technology. Which technology you use is usually dependent on the questions you’re trying to answer – and how fat your wallet is….
Anyone who has spent any amount of time in a cell or tissue culture lab will have experienced contamination at some point. You might not admit it, or want to admit it, but you know you have! I performed my graduate work in a basement university lab with an out-of-service emergency exit door in the…
Need to stain Gram-negative organisms? You should consider the Warthin-Starry stain.
Parents of small children attending nursery know that the period of time from September to June is a succession of colds and flues for the whole family – children with their underdeveloped immune system exchange viruses, creating new potent strains. Well, that’s probably how bacteria feel all the time in the natural environment teeming with…
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 your…
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.
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. …
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…
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)….
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