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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.
Here’s an all-too-often repeated scene in the lab: First thing in the morning, you approach the 37°C incubator with trepidation, open it and through one half-open eye you take a look at the LB plate that you spread your ligation-reaction-transformed E.coli aliquot onto. Looks good – thousands of colonies. Emboldened, you take your “no ligation…
Sepharose beads are porous, which gives them a high surface area for interaction with proteins and allows them to hold a lot of liquid. This is perfect for the application that they were originally designed for: purifying milligrams of protein in columns. When immunoprecipitation (IP) – a small-scale technique for pulling specific proteins out of solution using…
Virtually every research scientist has a use for sterile technique in the lab, whether you study infectious microorganisms, do tissue culture, or use E. coli for cloning. Good sterile technique is a basic lab skill required to avoid contamination of your materials and experiments; and fortunately, the principles are simple to learn and easy to…
At the end of my last article, I provided some practical tips and tricks for working with enzymes at the bench. Now, we’ll cover one of the cornerstone techniques of enzymology work: the enzyme assay. Starting with the simple assays and eventually working our way to the more complex, this article introduces the principles of…
A partial digest – typically done when you only want to cut one of two or more restriction sites in a DNA – can be a frustrating procedure to execute. The best advice anybody can give about partial digests is to avoid having to do them. However, there are times when there just aren’t many…
Western blotting is a common lab technique used to detect and analyze proteins. It also happens to be a really long and complicated procedure, with many steps along the way that are easy to mess up. How do you make sure that your Western blot is successful? Avoid the following five ways to destroy your…
In Part 1 of this series, we began our journey into the fascinating world of enzymology. We looked at the most basic concepts of what an enzyme is and the incredible jobs it can do. In Part 2 of “Working with Enzymes,” I will look at some things that you should keep in mind to…
In parts one and two of this series I described how semi-permeable membranes and precipitation methods could be used to concentrate your protein-of-interest, but there is one more method that you may not have thought of for protein concentration – chromatography. While chromatography resins are an obvious choice for protein purification, they can also be…
While precipitation is an obvious choice for concentrating DNA and RNA samples, it can also be an effective way to concentrate proteins. Here in installment two of this three part series, I describe the two most common methods for protein precipitation – ammonium sulfate and trichloroacetic acid. Background Precipitation of proteins occurs primarily by hydrophobic…
This is the first of a three part series describing some of the most common methods for concentrating proteins. In later installments I’ll discuss using protein precipitation and chromatography to concentrate a protein. However, here I’ll detail the most popular approach – semi-permeable membranes, used for both dialysis and commercial protein concentrators. Structure of the…
Protein kinases and protein phosphatases phosphorylate and dephosphorylate a plethora of proteins. They are responsible for regulating the majority of cellular activities. Because of their importance, they can seem intimidating to tackle as a research project. At the end of the day however, kinases and phosphatases are- simply put- enzymes. Therefore, you can standard enzyme…
If you purify proteins expressed in E. coli, then you’re probably familiar with this scenario: you come in bright and early in the morning and inoculate your large flasks of media with the overnight culture, start shaking them at 37 °C, and now you wait. And watch. And wait some more. You can’t venture far,…
I learned most of my molecular biology skills in the first lab I worked in almost 10 years ago. I realized recently that I was in desperate need of a refresher course, so I did a little bit of reading to see if I could improve the efficiency of my cloning reactions. In the process,…
Chris Dieni wraps up his series on the Basics of Protein Phosphorylation with this final part on further reading and resources available.
Chris Dieni explores the promiscuous yet intriguing nature of protein phosphatases
CIP has been around for a while, but there is a better alternative.
Using UNG is a great trick for PCR amplicon decontamination, but is it any good for rtPCR?
Diving deeper into protein kinases, explore the vast realm of known kinases, their amino acid targets, and the intricate process of target selection.
If you regularly grow up the same bacterial culture, whether it’s the strain that expresses your favorite protein, the culture you make your competent cells from, or just your regular control strain, it can be a bit of a pain growing it up from scratch each time. Before you even get to grow your actual…
In the previous article in this series, we looked at the major players involved in protein phosphorylation: protein kinases, protein phosphatases, and target proteins. This time, we’ll glance over some of the tools that we can use to study various aspects of protein phosphorylation, focussing on a few I’ve personally come across. 1. Tools for…
Primer design can sometimes feel like more of an art than a science, and designing the best primer can significantly affect the success or failure of your experiments. Here are a few tips on optimizing primer design for several different applications: PCR amplification/cloning One of the most common primer-based applications is cloning. The desired amplicon…
The focus of my grad studies and postdoctoral research has been the analysis of proteins regulated by reversible protein phosphorylation. However, the number of unique facets in which protein phosphorylation can be studied is astounding, and is diverse as any niche of the biosciences. This article is the first in a series that will give…
What is HRM Analysis? It is now more than a decade since the introduction of melting analysis to characterize PCR products. Melting analysis following SYBR green-based real-time PCR has become a mainstay in research laboratories worldwide for applications such as gene expression because of it’s ease of design and cost-effectiveness (i.e. no need for expensive labeled probes)….
We recently featured an article about how to streamline your cloning. But what about those days when you have too much on your plate, and need to put some things off until later? Here are a few hints on where you can pause in your cloning experiments while working on other projects: We sent a…
Northern and Southern blotting are now a thing of the past. They’ve been replaced with a faster and more quantitative technique. No longer do we wait days to know whether a gene is expressed. We can have the answer in 45 minutes! Real-time PCR is now commonly employed in almost all molecular biology laboratories to…
One of the very first things you need to do when getting set up for quantitative PCR (qPCR) is to determine the efficiency of the assay because knowing the assay efficiency is critical to accurate data interpretation. Here’s how.
I always keep an ear open for helpful tips in the lab – those little tricks that can make your experiments faster, easier, and better. Here are a few tricks I’ve picked up for trimming down the time it takes to do your cloning. Restriction digests Many digests are complete within 10 minutes of digestion…
PCR is highly sensitive, but the downside of that very property is that it makes the technique prone to producing false-positives. In labs where PCR is a staple, like the one I work in, any false-positives are more often than not due to amplicon contamination. A broken capillary or a PCR plate left carelessly at…
If you have ever attempted to load a SDS-PAGE gel only to miss the well, stab the divider, and then watch helplessly as your sample squirts off into the wrong well, then this tip is for you. The fortunate among us are able to use pre-cast gels with the wells outlined on the gel plate,…
We received the following question from Bitesize Bio reader, Beheroze Sattha. It relates to a problem with absolute quantification using plasmids for standard curves. Since many people use this technique it is an interesting one question for us to explore, and it also gives us a great opportunity to cover some important tips for performing…
If you’re performing DNA/RNA precipitations, you will have read Suzanne’s excellent article on which alcohol to use for precipitating your precious samples (check out some useful info in the comments for that article as well). Its publication prompted the recall of a useful tip I learned from a post-doc many years ago, one of those…
There’s something in the water, and it would love to go after your experiments. Straight out of the tap, water contains microorganisms, endotoxins, DNase and RNase, salts and other impurities that could gobble up your experiment in one bite. Of course we avoid this drama completely by using purified water from which these nasties have…
Working at a plasmid repository, I get a lot of feedback from scientists who are relieved we exist simply because that means they don’t have to request a plasmid directly from another academic lab. Either they’ve had a bad experience making requests in the past, or they really don’t know how to go about doing…
TAE or TBE, which is best? Well, of course, it depends on what you want to do. Here are the pros and cons of both: But you might be better of using neither of these buffers. Despite the fact that they have been firmly established as the most popular buffers for DNA electrophoresis since their…
One of the great features of PCR is its excellent sensitivity as we know. And many articles describe real-time qPCR as an added leap forward in that sensitivity – to the point where it has become a standard feature of a new assay description Indeed, I’m currently developing a new qPCR assay to replace a…
If you’re like many researchers, problems with PCR amplifying high GC DNA templates will be a major annoyance for you. Many strategies developed to overcome this issue. Betaine is the most common PCR additive used to enhance amplification of GC rich sequences because of its ability to dissolve secondary structure that blocks polymerase action. But…
Designing effective qpcr primers is essential for successful qPCR experiments, yet it can be challenging. Many validated primers are available through commercial databases like Qiagen’s GeneGlobe and Applied Biosystems’ TaqMan assays. Alternative public databases also offer primers, mainly for human and mouse genes. When primers are unavailable, literature searches or custom design remain necessary. Using pre-designed qpcr primers can save time and improve assay efficiency, making them valuable resources for bioscience researchers.
It’s the molecular biologist’s version of ‘I have good news. . and bad news’. The good news is that I amplified the DNA band of interest. The bad news is that I amplified these other bands as well! Oh, and this smear. What to do? Typically you might try and cut out the band of…
Hydrolysis probes are a widely used chemistry in RT-qPCR for sequence-specific detection through probe hydrolysis and fluorescence increase. Compared to PlexorTM, they offer equivalent signal specificity but lack melt curve analysis. PlexorTM provides easier optimization and direct signal measurement, while hydrolysis probes require careful probe and primer design for accurate results.
This article provides a clear comparison of Plexor and SYBR chemistries used in qPCR. Key differences include signal specificity, PCR inhibition, melt curve analysis, and multiplexing capabilities. Plexor offers greater specificity and multiplexing potential, while SYBR Green is simpler but prone to non-specific signals and PCR inhibition. The article also discusses some limitations unique to Plexor. Overall, Plexor and SYBR compared highlight practical considerations for selecting qPCR detection methods.
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