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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.
Got non-specific PCR amplification? You need touchdown PCR! Discover what it is, how it works, and get 5 top tips for performing touchdown PCR.
Several years ago as a freshman in a research lab, the very first project I received was to pipette incremental micro-volumes of H2O onto a piece of parafilm. Boring! Weighing the liquid on parafilm and comparing the weight between 10 replicates for each micro-volume continued for a week before I touched anything else in that…
Using UNG is a great trick for PCR amplicon decontamination, but is it any good for rtPCR?
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…
Find out how to build a plate centrifuge using a salad spinner. Gathering the components is as complicated as it gets!
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.
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…
In my last article, I introduced Cell-Free Protein Synthesis. Today I want to talk about a major bottleneck in in vitro cell-free protein expression; low yield. Most often, paying attention to the important factors such as template purity and design, transcription and translation inhibitors, and potassium and magnesium concentration will solve any problems with low…
Cell-free protein synthesis (aka In vitro translation) refers to protein production in vitro using lysates generated that provide the cellular machinery necessary for synthesis. The lysates can be of bacterial or eukaryotic origin. It is a useful alternative to in vivo synthesis for generating protein for the study of things like: So what are the…
qRT-PCR (quantitative reverse transcription-polymerase chain reaction) is now the gold standard technique for mRNA detection and quantification, sensitive enough to enable quantification of RNA from a single cell. The reverse transcription (RT) step is the main source of variability in a qRT-PCR experiment, so an optimal reverse transcription is essential for a reliable and successful…
Transfection of eukaryotic cells is a routine but sometimes tricky procedure. There are several transfection reagents available on the market, but sometimes the old methods are the best. I find that the simplest, fastest and cheapest transfection method for eukaryotic cells is calcium phosphate mediated transfection (1). It’s main advantage is that, since Ca2+ is…
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