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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… Did my electroporation work? Is my vector inside my competent cell? Do I have contamination in my liquid culture? Is this the correct bacterial strain the neighboring lab promised me it is? Did the guy from the other side of the world send me the…
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.
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…
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 –…
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: Expensive exonucleases, called restriction enzymes: pacman-like enzymes that chomp at specific sequences in your destination vector or fragments to be inserted (often just “inserts”). Sequence homologies between your inserts and your destination vector, called…
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…
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…
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…
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…
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…
You are probably familiar with fluorescent in situ hybridization (FISH) to detect and localize the presence or absence of specific DNA sequences on chromosomes. But did you know there are numerous FISH experiment variations? Including high-resolution FISH and quantitative FISH? Read here about Fiber-FISH, Q-FISH, and Flow-FISH and decide if you would like to undertake one of…
If you are cooking up a way to test if two proteins interact, you need a yeast two-hybrid (Y2H) screen in your recipe book. You don’t want your Y2H to turn out half-baked – so check out this guide to Y2H and we’ll help you make sure yours will rise to the occasion! What is…
If you are establishing a flow core facility or even setting up just one cytometer, then you need to know a few things before you plan your new lab and start looking at purchasing cytometers: Before You Start 1. Know Your Customer It’s really important to know if you are going to be setting up and…
Want to measure how much mRNA you have in a particular sample? Easy! Make some cDNA, add some fluorescent DNA-intercalating dye, pop it into a quantitative real-time PCR (qRT-PCR) machine and Bob’s your uncle! You have your result! Easy right…? Not so fast. As with any scientific assay, qRT-PCR requires some optimization. First, you need…
Would you eat your spaghetti dinner without a plate? No, of course not! It would make a big mess and be ugly to look at. Instead you NEED something to put that spaghetti on, to contain it, to keep it clean, to make it look nice – a bowl, a plate, SOMETHING! By the same…
It was not long since the commercialization of NGS (a little more than ten years ago) that scientists went beyond the basics and got creative with the new technology to study much more than just the sequence of DNA. In this article we highlight some of the different NGS technologies and methods available out there….
Science is an expensive business and those who use high energy-demanding techniques may not even realize just how expensive they are. The Cost of PCR Let’s looks at PCR. You need to pay for the machine, all the ingredients including expensive enzymes, a freezer and a fridge for your ingredients, tubes and caps, not to…
Dear Aunt Yersinia, A very annoying postdoc in our group keeps telling me off for spinning E.coli at 13K in a tabletop centrifuge. The postdoc claims that high speed damages cytoskeleton and this will reduce my transformation frequency. But I don’t believe her as the cells are cushioned by water during centrifugation. Can you tell…
No matter how we make measurements, there will be variation (a spread of data). Take 100 people and ask them to guess your age and you will get a range of results: some will be too low (excellent!), some too high (not so good!). It is the same with any of our laboratory experiments –…
Plasmid incompatibility could ruin your experiments before you’ve even started. And if you don’t know what it is, you can’t troubleshoot it! This article explains plasmid incompatibility, so you can’t be caught out by it.
Labs across the world spend a great deal of money on Taq polymerase. Find out how to save your lab some money.
“Making the simple complicated is commonplace; making the complicated simple, awesomely simple, that’s creativity.” – Charles Mingus Next Generation Sequencing (NGS) technology has boomed in recent years, allowing researchers to probe further into the workings of the genome. According to the theory of simplicity, it is the simple principles on its basis that make…
Problems with expressing your gene? One of the potential stumbling blocks in heterologous gene expression is incompatible codon usage. Every amino acid can be encoded by more than one codon, and for every amino acid each organism has a favorite codon that it tends to use more often than the others. The availability of tRNA…
If you’ve ever performed PCR, you’re probably already very familiar with DNA oligonucleotides (or oligos). But did you know that these molecules can do so much more than just act as simple primers? You can add a wide range of modifications to your oligos, which may change the stability, binding, solubility and even visibility, to…
Discover the different approaches to FRET quantification and get tips for selecting your FRET pairs.
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