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Think of the very first time you looked at cells under a table-top microscope. Here’s what you would have done in that experiment: Step 1: Grow cells. Step 2: Plate cells on to a glass/quartz slide. Step 3: Insert the slide under a microscope and look. The protocol for performing single-cell microscopy has a similar…
The ELISA (enzyme-linked immunosorbent assay) is arguably one of the most important and versatile tools in the toolbox of molecular biologists, biochemists and diagnosticians across the world. Defined by its simplicity and speed, the assay is easy to learn and perform in as few as five steps. But with so few variables to manipulate, an…
Let’s say you work with a gene, and it has wonderful potential. Excitedly, you throw your gene into the cells and voila! It’s there. Great. Now what? Genes are powerful tools for directing cell activity, but thanks to that curiosity characteristic of scientists, we want to know more: So what do you do? You had…
Last time we brushed up on cell referencing and constructing formulae to use Excel for some basic and some slightly more advanced calculations. This time we’re going to move on to using some built-in Excel functions and go through how to apply all that we’ve done so far to a worked example that is relevant…
Protein expression is an art. There are many routes to optimize a protein expression protocol, such as using different expression systems (e.g. E. coli, yeast cells, insect cells) or changing the expression vector or culture media for the expression host. Fortunately, optimizing the parameters mentioned above often leads to improvements in your protein expression results. However, there is one other…
Microsoft Excel can be a really powerful, useful tool for certain kinds of data processing and record keeping, and the chances are you probably don’t even know how to use half of the functions it comes with! That’s OK, personally, I find Excel a bit less user-friendly than Word, but also it’s a programme I…
Everybody’s talking about them, tens of millions use them, but do you know anything about optical fibers/cables? Well you should. In this first (of many) articles I will cover the basics of optical fibers, what they look like and what they are made of. Optical Fiber Anatomy First thing first, the terms “optical fiber” and…
Anyone who is remotely associated with any kind of drug development program knows how challenging the field is. Not only is the process complex and time consuming, but it also requires the concerted effort of experts from various disciplines such as chemistry, biology, microbiology, pharmacology, toxicology, etc. Whenever such collaborations are not possible, you are…
Next gen sequencing is a powerful technique, one that now lies at the heart of many scientific projects. This power comes with some special challenges, however, and by recognizing them you can ensure that your NGS results are robust. No one wants to publish findings that other scientists fail to replicate, but unfortunately it happens…
Applying molecular techniques to unicellar organisms leads to many questions… All these questions can be answered by going through the slow, routine pipeline process in a molecular biology laboratory: Culture cells, harvest them, extract DNA and PCR. Or guess what?! You can directly perform PCR using the colonies on the petri dish that the post-doc…
What is cDNA? …and how do you choose the right tissue to make a cDNA for isolating your gene of interest? Here’s what, and how.
You’re applying for your first tenure-track position, and you’ve heard that your dream department uses something called the h-index to decide who will get interviews. It’s an increasingly common scenario: institutions are now regularly using the h-index to help make hiring and promotion decisions, especially when they have to screen many applicants. For that reason,…
A Brief History of Detecting Amplicons The Old Days of Ethidium Bromide In the early 1990s, quantitative (q) PCR was in its infancy, and despite PCR itself already being around for 10 years, there were no easy ways of precisely quantifying the amount of DNA that was amplified in a PCR reaction. In those days PCR…
After panel design, and titration of the reagents, the next most important step in flow cytometry is setting the proper voltages on the photomultipler tubes (PMTs). These detectors take the photons of light emitted by the fluorochromes on the cells and convert them to electrons, which ultimately become the voltage current that is digitized and…
Western blotting can often be a source of frustration in the lab. Getting a beautiful Western is hard work, and it’s even more difficult when trying to visualize large molecular weight (>150 kDa) proteins. Here are five tips you can use to get a great blot: 1. Choose the right gel composition With all of…
When I first started out in the lab, I used to follow all protocols to the letter. Now, this is fine as long as your reactions run smoothly. However, there came the day when I had a new protocol and I just couldn’t get it to work. My supervisor told me to just “play around”…
Most of us use pretty standard transformation protocols for E.coli. Yours probably goes something like this: – Thaw the competent cells on ice– Add DNA– Electroporate (or incubate then heat shock for chemically competent cells)– Add rich medium (LB or SOC)– Incubate at 37°C (or appropriate temperature) for 30-60 minutes– Spread onto antibiotic plates That…
When you think about separating proteins, do you think about separating them using a gel? Specifically using SDS-PAGE? If you answered “yes”, it is for good reason. SDS-PAGE is ubiquitous in molecular biology labs because it is good at separating proteins. However, SDS-PAGE takes a lot of time and is labor-intensive. So let’s expand your…
If you use a Becton Dickinson (BD) cytometer in your lab, the chances are you are acquiring your data using ‘Diva’ software. Diva software is used to acquire your cytometry data on LSRII, LSRFortessa, CantoII and Aria cell sorters. As well as acquiring your data using Diva software, you can also analyse your data after…
Microscopy is one of the fun parts of working with yeast. If you fix your cells, you can get a snapshot of the structures. Live cells microscopy using fluorescent proteins tagged proteins is even better, as you can see the dynamics and cell machinery working before your own eyes. Light Microscopy to Check for Contamination…
Traditionally, if you’re hoping to clone a DNA/RNA fragment (or insert) into a vector, such as a BAC you would need: This process looked something like this: cut you vector using exonucleases to expose ends that are complementary to the ends of your insert and then stick your insert into the vector using a ligase….
An ad about a Technical Officer position is usually nebulous. For example: “The post holder work as part of a technical team and provide both routine and specialist services in support of undergraduate, postgraduate, outreach and revenue-earning activities.” What Does a Technical Officer Do? In fact, a technical officer role can be summarized in two…
If you were to peek into a protein biochemist’s bag of tricks, what would you find? A mortar and pestle for collecting samples, some columns for isolating proteins and a mass spec instrument? Perhaps. But what about those little eppendorf tubes full of enzymes and helpful molecules? Certainly, each scientist has his/her own favorite. Here…
Ever wonder why your data isn’t the same after repeating an experiment? Well part of science’s beauty lies in the difficulty of achieving reproducibility. Heraclitus first said that no mans steps in the same river twice and the same can be applied to experiments. It is literally impossible to control for everything because the second…
Generally, once you’ve acquired your samples on a cytometer, the hard (but hopefully fun!) part of making sense of all the data begins. This can be increasingly complex depending on the number of cells, populations, parameters, and combinations. So where we use the dedicated software aligned to the analyser for acquisition, we frequently use alternative…
For many, myself included, the idea of attending a large conference and talking to hundreds of new people can be terrifying. Often times there are people in attendance you may want to collaborate with, or that you see as possible future employers, and you really don’t want to make a bad first impression. Luckily, there…
In the cell culture practice, heat inactivation of serum products has always been accepted and is one of the basic protocols passed on to new cell culturists. There is no strict standard protocol for heat inactivation, some say incubate at 56 °C for 30 minutes, while some say it can be efficiently performed using temperatures…
Using fluorescent proteins as imaging probes is a widespread and versatile technique in microscopy. You can use them in a wide range of living systems, from single cultured cells to complete organisms and animals. Fluorescently tagged proteins can be used to track and examine real-time localization, interactions, and translocation of your protein of interest, as…
The recent advancement of next generation sequencing technology and the development of novel gene editing tools, such as CRISPR-Cas9, have revolutionized research in genetics. In this golden era of molecular biology, knowing how to dig and navigate through all the enormous sequence information is an essential skill for most molecular biologists. However, to obtain facile…
PIPE PCR is a ligase-independent, restriction enzyme-free cloning strategy like SLIC (link to my SLIC article), SLiCE and CPEC. The PIPE method eliminates sequence constraints and reduces cloning and site mutagenesis to a single PCR step followed by product treatment. It is fast, cost-effective and highly efficient. The key step is designing the primers; one…
Mycoplasma is one of the biggest threats to cell culture. It’s quite easy to test cell cultures for the presence of mycoplasma using PCR, and we’ve got a simple protocol for routine testing.
In part 1, “The Power of STED Microscopy: How Does it Work?” I covered the basics of STED Microscopy: how it works and why you might want to use it. If you now want to do STED Microscopy, the next step is to optimize your sample. In this article, I cover how you can get…
Recently we wrote an article about widespread cell culture contamination and how to detect it. This follow-up article will provide practical tips on avoiding cross-contamination in the first place. Be Cautious While Working The first way of cross-contaminating cultures is by accidentally mixing two cultures together, which may lead to an unintended co-culture or the displacement…
Part of my job in running a core flow cytometry facility is to make sure that the experiments that my users run have been optimised. But that optimisation can be split up into several areas. The first area is experimental planning: What do you want to know? Can you do this by flow cytometry? And…
While working in a UK university, I met a researcher who loved Italy much more than the UK. I asked her why she had left her favourite country. She told me that before coming to the UK, she had a 2-year fellowship in Italy where she was getting some promising results and had the chance…
PCR contamination can be your worst nightmare if you are not able to figure out the source of the bands in your negative control. Initially, when I had to setup PCRs, contamination was my main problem and it remained a mystery for a while, as no matter how many times I repeated it with a…
You’ve got an advisor, you’re done with classes, you’ve finally passed your qualifying exams and your dissertation project is underway. Life is looking good, but it’s not too early to start thinking about how to tackle your dissertation. Chances are this is the biggest writing project you have ever undertaken, so breaking it up into…
If you want to see in real time what is going on inside your cell then you should be performing live-cell imaging. Live-cell imaging techniques allow real-time examination of almost every aspect of cellular function under normal and experimental conditions. With all live-cell imaging experiments, the main challenges are to keep your cells alive and healthy…
You don’t need to be told about how next generation sequencing technologies have revolutionized the way we study the genome and the epigenome. Whether you want to look at transcription (RNA-seq), translation (Ribo-seq) genomes (DNA-seq), interactions of proteins and DNA (ChIP-Seq) or to study epigenetic features such as methylation (whole genome bilsulfite sequencing) there are…
If there is a job in the world that requires one of the highest levels of motivation, I would say it is doing a PhD. Pushing yourself out of bed daily to enter the lab ain’t an easy task, especially when your results are dodgy or you have unluckily found that your lab-mates or project…
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