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T cells can be problematic to characterise because they have a wide variety of subtypes and because of the technical difficulties of studying the membrane-bound T cell receptor, but there are situations where you want to be able to do this such as analysing the degree to which immunological memory has been induced to measuring…
“It is the weight, not numbers of experiments that is to be regarded.” Isaac Newton Read any flow cytometry protocol and somewhere near the beginning will state something to the effect of ‘Place 1 million cells into a tube.’ The question is, faced with that special sample for THE experiment, how do you count cells…
Bacterial cultures may be much easier to grow than mammalian cells, but if your yields are suboptimal there are plenty of parameters to play with. Here we list a few of the things you should consider to maximize your culture growth. Shaking speed Shaking is performed to allow aeration of your culture, which is of…
Having kids changes your life. I should know, I have 5 little F1’s running around. Your life is thrown into chaos the minute you hear that first cry. And it isn’t only your personal life that changes. Eventually you have to figure out how to fold your new parenting responsibilities into your lab life….
Chemical chaperones are necessary in protein experiments. From buffers to storage solutions, chemical chaperones silently make proteins happy and soluble. Read this article to appreciate the love that chemical chaperones bathe on your proteins and learn when to use them! A chemical chaperone is a molecule that promotes the favorable interaction of protein with water…
Large amounts of data? Check. Repetitive tasks? Check. If you work with next gen sequencing data, you have probably already realized it’s a good idea to learn a scripting language. But learning a programming language is a major endeavour, and with lots of languages available how do you decide which one to study? And once…
If you want to make molecules stick together you need to know about streptavidin/biotin. This article follows on from Mike’s article looking at ‘sandwich’ and ‘amplification’ methods of immunohistochemistry (IHC) and covers how streptavidin-biotin works in IHC, including protocols. Streptavidin-Biotin What is it? Avidin is a natural biotin-binding protein found in egg whites. Streptavidin is similar…
As Morrissey once sang “in the midst of life we are in death, etc.” A fact of life in the research Lab is that whenever we run an assay there will almost certainly be some cell death in our sample. This may be insignificant in some techniques but in others it can be problematical. Why…
Have you ever been shown how to use a microscope properly? Or do you just dive right onto the microscope with little or no training and scant knowledge of the basics, then twiddle knobs, snap photos and expect the publication-quality images to appear? If it’s the latter you are certainly not alone! If only there…
When I buy a new sweater, I love finding out that it goes with several pairs of pants, the scarf that’s an awkward color and the earrings I haven’t worn yet. PCR is like this sweater – it goes with almost everything and molecular biology is taking full advantage of this using it at every…
Making a Next Generation Sequencing (NGS) library can seem a bit daunting to the new user, as failures can be expensive. But don’t be put off, as NGS library preparation is relatively simple molecular biology, and can be very easy if you choose to use a commercial kit from one of the many suppliers. Take…
The biggest source of PCR contamination is aerosolized PCR products. How do you fix PCR contamination and avoid it in the future?
My PhD is rapidly becoming a distant memory. Before nostalgia completely obscures my recollections of this chapter of my career, I thought I’d jot down some pointers for prospective and current PhD students. These are mainly based on things I wish I had done during my PhD, or mistakes I have seen others make. I…
I often wonder why it is that molecular biology researchers stubbornly refuse to change 40-year old methods that, while work, are not as good as newer, faster and cheaper methods out there. I suppose rational scientists often have irrational superstitions. One example of an old method that could be improved is the growth media used…
Gene expression analysis plays a pivotal role in a wide range of studies, including biomedical analysis and diagnostics. Of all the methods available for gene expression analysis, quantitative real-time PCR (qRT-PCR) is the most rapid, sensitive, and accurate to measure mRNA, and its use in clinical diagnostics is rising steadily. RNA quality entails both purity and…
No matter how efficient you are, it’s always possible to improve your productivity and improving your productivity means that you get more of the rewards you are trying to obtain: results, publications… or dare I say it, money. Here are 20 ways to improve your productivity. Some are focussed toward improving the productivity of bench…
The Rise and Fall of the 454 Sequencer The GS20 454 sequencer, released in 2005, was the first next-generation DNA sequencer to hit the market, and its feats quickly dazzled the scientific community. As new sequencing platforms proliferated, however, many researchers opted for less expensive options and 454 market share fell. About a year ago,…
When you fix your tissue samples with paraformaldehyde (PFA) the proteins in your sample become covalently cross-linked. This is good to preserve the ‘architecture’ of your tissue sample. However, this cross-linking can become a problem when you carry out immunohistochemistry (IHC). Cross-linking can ‘mask’ or hide your antigens-of-interest and make them ‘invisible’ to your IHC…
The half-life of a protein is an important factor in many molecular biology studies. If your thesis has anything to do with proteins then your graduate advisory committee will ask about half-life, so start planning your 35S experiment today. To help you here is my overview of the major steps of 35S labeling complete with…
If you are bringing cells to a core to be sorted there are a few things you can do to optimise your sorting and will help the core staff. Here are a few hints: 1) Know the size of your cells Do you know the size of your cells? Not many people do and its one of…
Mercury burners are one of the most common light sources used in fluorescence microscopes, producing a wide spectrum of wavelengths making them a great light source for viewing your samples. But they do have their issues – one of which is their propensity to break or become damaged if not used and cared for correctly, which…
Today, PCR is as common a feature to the lab as pipettes and beakers. The majority of us regularly need to amplify our DNA or RNA samples, sometimes for an ‘everyday’ PCR run just to check if our primers actually work, or in a quantitative (q)PCR run, where we might be comparing the levels of…
Figuring out what’s what When studying cells and cell subsets (and cell sub-subsets, and so on!!) we need ways to identify and classify every single cell. This will allow us to individually analyse each population and, for example, help to discover their role in health and disease. A principal way we do this is by looking…
Do you need purified recombinant protein to test biological drugs? But you don’t have the time or facilities for cell culture and purification? Try using in vitro translation (IVT) instead! IVT is like your own miniature protein factory. And it is great for a wide range of molecular biology applications because IVT can be much…
Today I would like to introduce you to pUC18, a plasmid most noted for its high copy number. In the first article in this series, we talked about how origins of replication (ori) control plasmid replication and copy number. To learn about this, we focused on the pBR22 ori and the role of Rop protein in…
SLIC, or sequence and ligase independent cloning, was developed by Li in 2007 and published in Nature Methods. What makes it a Nature Methods worthy protocol? Unlike other forms of cloning, SLIC does not require restriction enzymes or a ligase! Seriously! Don’t believe me? Why not have a go for yourself? I’ve detailed the main steps below to get you started. How it works To…
When you’re trying to solve a PCR problem, you’ll probably resort to a Google search at some point or another. Here’s a list of the Top 10 go-to websites to help solve your PCR problem.
Power nap anyone? Depending on how long your commute is, and what type of transport you use, you could make your commute useful. If you are taking public transport, you can use that time to answer those emails you don’t have time to get to at the office/lab, or to catch up on reading some…
You’ve nurtured your cells for weeks, perfected your experimental conditions, and nailed down all the controls. You’ve harvested your cells and gently lysed them, now you’re ready to look at the proteins. What’s one of the most common next step in protein analysis? A denaturing gel or SDS-Polyacrylamide Gel Electrophoresis! SDS-Polyacrylamide Gel Electrophoresis, or SDS-PAGE…
Do you suspect that your favourite protein is doing something really cool? But you cannot see it because your confocal microscope’s resolution is limited. Then Stimulated Emission Depletion (STED) microscopy is what you need! With the power to smash through the diffraction limit of confocal microscopy, STED opens up a whole new world of improved…
Copyright is something that a lot of scientists only give a passing thought to. However, this is something that affects us all. If you publish your work, then you need to understand copyright, the different types of copyright, the difference between open access and copyright and what you can and cannot do under different copyright…
A lot of chemical reagents are relatively non-hazardous.But there are just as many that are extremely hazardous, which means you’ll want to take precautions to reduce any risk of exposure, repeated exposure, and of course, accidental contamination of anything – or anyone – that walks out of the lab at the end of the day….
Working in a lab in a developing country can be a unique and exciting opportunity for any scientist. It can be very rewarding, but also challenging as you navigate foreign settings to conduct your research. Here are ten tips for working at the bench in developing countries. 1. Expect cultural differences Everyone approaches science differently…
Oligonucleotides are those smallish bits of DNA or RNA that we rely so heavily on for many of our molecular biology experiments. In their naked form, they are single, inert strands of DNA or RNA bases. But if you dress them up, you can increase their functionality. Here are some of the common oligo wardrobe…
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…
Over the years, I’ve had my fair share of ups and downs in the lab. The latter quite often centering on a failed or plainly weird PCR experiment. As I’ve gone on and become ever more fastidious about my lab practices I’ve realized that the majority of these little calamities were perfectly avoidable. In my…
Every protein is unique and thus every protein has its own set of production and purification challenges – many of which cannot be predicted. Therefore to successfully produce and purify your favorite protein you need to know and understand these five unpredictable protocol variables (X factors). Tweaking these X factors just might be the difference…
Colocalization blues (and reds and greens) Trying to find if and where two epitopes co-localize (or, to be more precise, where they are found in close proximity) may seem easy at first: 1) Bind your two epitopes with primary antibodies from two different species, 2) bind these primary antibodies with two secondary fluorescent antibodies, one…
A marriage of sorts Fluorescence resonance energy transfer, or FRET, is often done using a microscope, which means it can be difficult to analyze large numbers of cells in one sitting. One way to overcome this, is by combining FRET with fluorescent-activated cell sorting (FACS), giving you a high-throughput method to screen for protein interactions…
What do cell lysis, clean dishes, and gallbladders all have in common? Answer: detergents! These useful chemicals can solubilize fats and other proteins in water. They are the key to applications as varied as lysing cell membranes, extracting DNA, and solubilizing proteins for gel electrophoresis. To help you understand these important chemicals, we provide a…
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