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You might think Northern Blots are an old-fashioned technique. However, qRT-PCR is prone to false positives and negatives, and reviewers may require Northern Blot confirmation of your qRT-PCR results. So sometimes Northern Blots are a necessary evil.
The development of CRISPR/Cas9 technology has made it relatively straightforward to selectively edit genomes and has revolutionized the way in which we approach biological questions. CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats and in simple terms, this technique allows you to direct a nuclease to cut at a specific site in the genome of interest. The…
Formalin fixed paraffin embedded (FFPE) tissues are valuable samples that typically come from human specimens collected for examination of the histology of biopsies for the detection of cancer. But each sample contains much more information just waiting to be unlocked. Despite the tiny sample size, DNA can be extracted from the tissue sections and used…
Hope you had a heavy breakfast, because the first qPCR is going to take time. Did you remember to fill your coffee cup? There are a lot of intricacies in qPCR that will need your neural networks to be brisk. How qPCR differs from traditional PCR Unlike traditional PCR, qPCR measures the amplification of DNA…
Are you an assiduous biologist who prefers label-free imaging methods for biological samples analysis? Raman spectroscopy offers you a wonderland of imaging technique with unlimited benefits. To start with, Raman Spectroscopy is a spectroscopic technique based on inelastic scattering of monochromatic light usually from a laser in the visible or near infra-red part of electromagnetic…
When choosing a model system for culturing tumor cells, we often think of the obvious tried and tested models. In vitro methods include cell lines that have been specifically selected to grow in culture flasks in incubators. While conveniently available, consistent and reproducible, cell lines are limited in that they may not represent the desired…
While it is true that there are some useful websites like SNPedia, or NCBI that can help you find rs codes for genetic variants, sometimes you need that info coming straight from the oven – particularly when you want to look at atypic SNPs or substitutions that have not been validated. So, in this post I…
We have to rely on artificial systems to test our hypotheses and often have to come up with original set-ups to investigate specific problems. One of these creative inventions is the use of supported lipid bilayers.
We’re already gone through the basics of how gel electrophoresis work, compared common gel types like agarose and polyacrylamide and even explored some alternatives. Now let’s look at the native versus denaturing gels. You’ll be a speGEList in no time! Denaturing Gels We’ll start with this one, as it’s very self-explanatory. Denaturing gels are exactly…
In real life, cells are instructed to commit suicide for the greater good of the organism. The programmed cell death (apoptosis) is important during development of a multi-cellular organism. A good example you will appreciate is the dis-appreance of the tail from a tadpole as it turns into a frog. On the reverse, the lack…
First of all let me say the technique of labeling tissues (immunohistochemistry, IHC), and cells (immunocytochemistry, ICC) is indeed immunoscience NOT alchemy, though at times it may certainly seem like alchemy! But to scientists inexperienced in this technique, who typically see the results of IHC/ICC experiments in the form of pretty pictures, it can certainly…
The first thing you learn about culturing cells is proper aseptic technique and avoiding contamination. After that you’ll learn all the ins and outs of culturing your project’s specific cell line(s). What may not have been covered, is co-culturing, and I don’t just mean ethnic diversity in the lab! Co-culturing is the indirect or direct…
If you’ve read our article, An Overview of Yeast Two-Hybrid (Y2H) Screening, you’ll know that one major limitation of conventional Y2H is that your protein-protein interaction must occur in the nucleus for the reporter gene to be activated. So what do you do if your protein is a receptor tyrosine kinase? Or a G protein–coupled…
So you have a gene or protein that you think may be involved in migration or invasion and the next step is to embark on migration assays. These assays are useful for testing fundamental migratory processes, such as embryonic development, immune response, metastasis and angiogenesis. For a long time these have been an invaluable mainstay…
As a protein biochemist where bacteria were mere workhorses, imagine my surprise when I began work in a bonafide micro lab! I discovered that bacteria could be much fussier than my good ol’ cloning and expression friends E. coli DH5ɑ or BL21. One broth would not do for all, some even required blood! No, no,…
I’ve noticed that there exists some ambiguity about the different terms relating to homology. I’ll try to break them down here, with significant help from Genome Biology.
So, you’ve extracted your precious RNA and want to check its quality on a gel. Conventionally, you would run a formaldehyde gel, which is messy and requires a lot of prep. Plus, it is a huge undertaking in terms of time (and money) if all you want to do is just check the quality of…
Anyone working with laboratory animals has probably realized that simply putting two animals together does not always yield new offspring and reliable continuity of the animal line – unfortunately animal husbandry isn’t that simple! Of course, apart from making sure that the two animals put together are from different genders, there are a lot of…
Fluorescent tags are widely used for microscopy and expression studies – but it wasn’t so long ago that this everyday tool was unheard of. In this article we’ll talk about how GFP came to be, and what it means for you. Green fluorescent protein, or GFP, was first identified in a fluorescent jellyfish, Aequorea victoria….
The resolution of any microscope is related to the numerical aperture of the lens and the wavelength of light used to form the image, and can be calculated using Abbe’s law. This, however, is the ideal situation – the best case scenario. In real life, resolution must be defined in terms of contrast, and there…
For several decades, Ethidium Bromide (EtBr) was the molecular biologist’s default dye for DNA staining. Now, EtBr is being consigned to the history books. It’s time to have a historical look at where it all started.
Generating a DNA vector library of single and/or compound mutants for a target protein can be a daunting task. If you’re lucky and work in a well-funded lab, you might outsource this process via gene synthesis. Most of us though, need to do it the old-fashioned way. Traditional QuikChange™ Traditionally, there are many steps you…
We have already looked into the different types of viral expression systems and when you might use one over another in my previous article. But why would you use viral transduction over similar techniques like plasmids? Just a reminder: Transfection is a lab technique where nucleic acids or proteins may be introduced into cells. When…
The human brain autofluoresces—a funny thought next time you see a cartoon character with a bright idea and a light bulb over his head—but not so funny if you are attempting immunofluorescence analysis. But there are some significant advantages to using fluorescence detection over chromogenic methods. In this article, I will cover the advantages of…
We can, however, use special dyes or engineered proteins to bind to calcium, indicating where in the cell it is. The trick is determining which probe is most suitable to your cells and application.
Supported lipid bilayers are a very useful tool in many fields of cell and membrane biology. But how easy is it to make them? Bilayers can be made quite reproducibly, once you have found a reliable protocol! However, it can take some time to optimize your technique, so to increase your chances, make sure you…
The Gram stain is another commonly used special stain in the histology lab. Why use a Gram stain? The Gram stain is a type of differential staining technique which represents an important initial step in the characterization and classification of bacteria using a light microscope. It is named after a Danish scientist, Hans Christian Gram,…
Cell Penetrating Peptides (CPPs) are the Trojan Horse of cell biology – an innocuous peptide sequence with the special ability to carry virtually any cargo across the plasma membrane. If you have a special delivery that you don’t want to get lost in transit, CPPs are for you! CPPs are short peptides (typically 5-30 amino…
Ever wonder what those small colonies, like satellites, surrounding a larger E. coli colony on your LB with ampicillin plates were? Or why, when you picked that colony, it never had the plasmid you just transformed? Well, it’s because those satellite colonies are “protected” from the ampicillin by the big colony. Read on for more… Ampicillin…
IMS is a rapidly advancing technique that nicely blends the best bits of mass spectrometry with microscopic imaging. Learn more about it and how it could be applied to your work.
One of the key characteristics of cytotoxic cells (i.e. CD8+ T cells, natural killer cells) is the presence of pre-formed cytoplasmic lysosomal granules. These structures house perforin and granzyme; two molecules that are essential for the lysis of target cells. Upon effector cell activation, granules are polarized toward the target cell and the contents are…
If, like many people, you use fluorescent proteins to view your transfection efficiency or your CRISPR gene editing, you can also isolate cells with different expression levels of fluorescent proteins. Here are 5 ways to improve your sorting experiment.
Ion Torrent technology, when it was introduced in 2010, was one of several machines that promised to revolutionize genetics. These were benchtop machines that showed their prowess in quickly sequencing smaller exomes and other DNA samples (about 10-20 million bases per run, compared to Illumina HiSeq, which could read 250 billion bases in a run)….
Whether you’re already in the field or an undergrad looking to enter the scene, here are some great places to keep up to date with the latest news and trends in stem cells. Listen About It For auditory learners, or people that listen to music on their way to the lab, you could switch it…
One of the most crucial steps in any cloning procedure is the preparation of the vector. Get it wrong and your chances of success will be drastically reduced. The overall aim for a good vector preparation is to obtain a fairly concentrated stock of undamaged, fully digested plasmid DNA that is free from contaminants. Missing…
Viability PCR (vPCR) is a big step forward in PCR technology. Through the use of a simple pre-treatment of the sample(s) of interest using specific intercalating reagents, it is possible to neutralize the DNA of dead cells. As a result, only DNA from live cells will be amplified by PCR. Through the vPCR, it’s possible to…
In a previous article, we went over the basic understanding of the inner workings of a flow cytometer. It’s important to grasp the types of measurements that are being made and, perhaps more importantly, what measurements are NOT being made. For simplicity’s sake, we’re going to frame this discussion in terms of a classical flow…
The sub-G1 assay for measuring apoptosis is easy, rapid, reliable, reproducible, and cheap and is widely used. However, you need to understand the mechanics of the assay and the apoptotic process, otherwise you could over- or underestimate your apoptosis results!
Producing lentiviral or retroviral vectors is theoretically fairly straightforward. However, anyone new to viral vector work is usually confronted with vast amounts of confusing information. It seems that anyone who has ever made a lentivirus has their own protocols and is adamant that their method is the best one to follow. In reality, there are…
Want to save money on one of the most expensive steps of your Western blots? Then, use less antibody for significant cost savings!
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