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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.
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.
Plexor offers a unique qPCR method that measures decreasing fluorescence during amplification, simplifying assay design and enabling melt curve analysis without probes or dyes. It uses modified nucleotides and reversible quenching for sensitive DNA and RNA quantification, supported by free primer design and data analysis software compatible with common qPCR instruments.
So you have some blood stored in the -20 °C or -80 °C and you want to isolate RNA from the samples. If you wanted DNA, you would have many products to choose from. But for RNA, your choices are more limited. Obtaining RNA From Frozen Blood is Difficult Why is that? The reason is that most…
The following question was emailed to Bitesize Bio by Beheroze Sattha and I gladly took up the challenge, and I immediately knew the answer. Or so I thought. After delving extensively into Pubmed, Genes V (I know, I need a new version) and Molecular Cloning I have come up with an answer, but it is…
Thanks to Bitesize Bio reader, Muthu Arumugam for contacting us about some problems he has been having with restriction digestion and clean up of DNA. I have boiled his query down to four main questions that are pertinent for most molecular biologists, so I hope that Muthu and everyone else can learn something from my…
In response to my last article, The Taq behind PCR, one of our readers, Bonnie Barrilleaux, asked whether DNA could naturally survive at temperatures that would denature it. It also begged the question; how do proteins stay intact and functioning at these high (55°C and up) temperatures? It turns out, cells do a lot of…
Gel extraction — what could be easier? Now we have quick and easy gel extraction kits, we no longer need to use time-consuming old fashioned methods like electro-elution or “freeze and squeeze”. Thank goodness. When Gel Extraction Goes Wrong But even the simplest of procedures can go wrong. Maybe you were distracted, confused, or thought…
Controls are obviously extremely important when setting up experiments. Without them, a meaningful interpretation of the experimental results would be impossible. I say obviously, but in my previous job as a technical services scientist, you’d be surprised at how often I found myself talking to customers about the importance of controls. One customer commented, during…
Reverse transcription optimization is one of the most overlooked aspects of RT-PCR best practices. The RT step converts RNA to cDNA, and it is one of the largest and most easily underestimated sources of technical variability in RT-qPCR. The problem is that RT efficiency is never 100%. It varies across transcripts and is sensitive to…
Nobel Laureate Kary Mullis is generally credited with inventing the polymerase chain reaction, but his discovery owes a lot to a microbiologist who loved to travel, some refuted assumptions of what can live in hot springs, and a now-closed field station in Yellowstone National Park.Here’s the story. In the 1960s, Thomas Brock was a biologist…
Removing unwanted DNA from vectors is one of the most routine laboratory techniques in cloning. Check out our top tips.
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