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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.
Don’t be confused by Ct values! We’ll guide you through what they are, how to calculate them, and troubleshooting issues.
Need to add extra nucleotides to your plasmid or other DNA sample? Here’s how to use overhang PCR to easily add extra bases using primers.
Struggling with your cloning? It could be time to try blunt-end cloning. This easy introduction explains what it is, when to use it, and how.
You can build your own PCR machine using tools and equipment you can get easily and without breaking the bank. Well, what are you waiting for?
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.
Are you having problems amplifying GC-rich regions in your PCR reactions? Read this article for 5 easy tips to address these issues!
New to qPCR? Here’s a quick summary of one of the two most common analysis methods – double delta Ct analysis.
As is sadly the case in many experiments, site-directed mutagenesis (SDM) does not always work the way we would like it to the first time around. Here are a few tips to help you on your way when trying to troubleshoot a bothersome SDM reaction!
Researchers have relied on immunodetection techniques such as Western blotting, flow cytometry and Enzyme-Linked Immunosorbent Assay (ELISA) for years, but immuno-PCR is a relatively new method. By merging an ELISA with the Polymerase Chain Reaction (PCR), immuno-PCR provides extremely high levels of assay sensitivity. ELISA An ELISA is an assay in which a molecule is…
The primers for your gene of interest have finally arrived in the mail, and you’re ready to figure out whether your favorite gene’s expression level is elevated in those precious tissue samples. Only one small last step before you can proceed. Primer validation. This is a standard procedure where you run PCR or qPCR on…
Allele-specific expression can occur for various biological reasons, such as gene imprinting, or differential transcription caused by mutations, or single nucleotide polymorphisms (SNPs), or epigenetic alterations. Traditional end-point RT-PCR or qRT-PCR-based methods only detect overall levels of mRNA expression from a given gene rather than mRNA transcripts originating from individuals. If your project requires more…
Who amongst us hasn’t had the need for oligonucleotides in an experiment? It is a cornerstone in many procedures and techniques. Depending on the goal, it can be very hard to design just the right oligo for your experiment. Oligos must have the right length; the right amount of C-G, T-A; they can’t form secondary…
It often happens that you do everything right with a PCR. You have perfectly isolated template DNA, used sterile tubes and tips, used clean reagents, and said a quick prayer to the PCR Gods. And still, something unknown messes up your results. This unknown at work is generally a PCR inhibitor. Before you blame it…
PCR was actually one of the first lab techniques I learned as an undergrad. Despite being sometimes labeled as a pretty basic lab skill, PCR doesn’t always work as expected. This “fickle” success is due to small details or hidden hazards within the PCR workflow that can cause your seemingly uncomplicated experiment to fail. This…
The polymerase chain reaction (PCR) is the backbone of many lab techniques. In short, it allows for the exponential amplification of a specific segment of DNA. Through the use of primers encoding restriction enzyme sites, these amplified fragments are used in downstream cloning procedures, usually leading to the insertion of one, maybe two, PCR fragments…
Droplet digital PCR? It’s easy. Because we’re here to guide you through it. We recently introduced you to the principles of digital PCR technology and how it differs from qPCR. In a nutshell, digital PCR is an end-point PCR technology that divides a single PCR into a large number of partitions, and then perform PCR…
We all know that genes encode proteins that make up a living cell. However, the level and coordination of gene expression is really the key to the success of a living cell. One way eukaryotic cells (that’s us!) control protein expression is through addition of a methyl or hydroxymethyl group on the cytosine nucleotide. This…
There are many different methods and protocols on making your PCR run more efficiently. I recently came across an interesting PCR method called “nanoparticle” PCR. This method seems to attract a lot of attention, because it enhances a PCR by a few orders of magnitude. More interestingly, while the enhancement effect has been reported in a…
So, you’ve designed PCR primers to amplify your sequence of interest, and you’re ready to go. But unless you have a never-ending supply of template, polymerase, and a thermocycler with a gradient function—not to mention a hefty dose of time and patience—you probably don’t want to spend the next week finding the perfect conditions for…
DNA shuffling uses PCR technology in a very creative way. It allows you modify your protein to make a new protein you want. You can evolve proteins in microcentrifuge tubes on your very own lab bench. Isn’t that fantastic? DNA shuffling is also a very powerful technique for directed molecular evolution. W. Stemmer first used…
An essential step in mouse breeding is genotyping them to determine the genotype of every mouse in the litter. It is also useful to differentiate between various groups of experimental mice if any confusion arises. When genotyping, you will be hunting for the specific gene that you want your mice to have or a genetic…
In the 30 odd years since its invention, the polymerase chain reaction (PCR) has become the bread and butter technique of molecular biologists. The secret to its indispensability lies in its simplicity and versatility. Numerous variants of the technique have been developed; one of these, real-time PCR, has become the method of choice for quantitative…
Routine PCR? Let’s be honest, there’s no such thing. Even with the simplest PCR reaction things can go wrong, so you need to have a good checklist of ideas for PCR troubleshooting and rectifying the problem. Today I have brainstormed all of the ways I can think of to approach problems with standard PCR reactions….
There’s piloting a brand new technique for the first time. Then, there’s jumping through hoops trying to get an established lab technique to work. The former, in contrast to the latter, is expected to be fraught with hardships. Yet troubleshooting an old lab technique that isn’t working anymore, is frustrating at a whole new level….
The more experienced hands in your lab know that molecular biology is rarely just a journey from A to B. As a result, I’ve constructed this short workflow as an introduction to genomic molecular techniques.
RNA purification may be a common procedure in molecular biology but it is by far the one that people fear most. Why? Dreaded RNase. It’s everywhere… all over your bench and pipettes, and floating in the air, waiting for the chance to creep into your prep, shred your RNA into nucleotides, and ruin a day’s…
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…
The ability for DNA polymerase to copy a long stretch of DNA is becoming increasingly important. Why? It has to do with the advances in our sequencing technologies. Our next generation sequencing (NGS) technology requires the DNA polymerase to copy a long stretch of DNA (sometimes up to 50kb) as NGS is churning out genetic…
Before I get into today’s topic, please allow me to digress a bit and start with a few sentences that sum up the polymerase chain reaction (PCR); the grand-daddy of molecular biology. PCR, a method that is at the heart of modern day molecular biology discoveries, is a process that amplifies genetic material through our…
Say you just joined a lab and have been assigned your very own project to work on. As part of your new responsibilities, you have to breed and maintain the mutant (or transgenic) mouse line which you will be using for your experiments. An integral part of mouse genetics experiments is determining the genotype of…
How to Obtain a Purer PCR Product and Reduce Non-specific Amplification Unless you’ve gotten your hands on some miraculously specific primers, amplification of only your target sequence without non-specific amplification can be very challenging. Thankfully, a clever and surprisingly simple solution is at hand! A Quick Recap of the Basics In PCR, you design your…
I think we all have been through those my-PCR-product-didn’t-get-amplified days. Sometimes, playing around a bit more with the PCR conditions brings luck, or sometimes it doesn’t work at all. These days we have access to many different types of DNA polymerases, ultrapure and buffered nucleoside triphosphates, and other necessary starting materials in convenient concentrations; but…
PIPE PCR is a ligase-independent, restriction enzyme-free cloning strategy like SLIC (link to my SLIC article), SLiCE and CPEC. The PIPE method eliminates sequence constraints and reduces cloning and site mutagenesis to a single PCR step followed by product treatment. It is fast, cost-effective and highly efficient. The key step is designing the primers; one…
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…
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…
When you’re trying to solve a PCR problem, you’ll probably resort to a Google search at some point or another. Here’s a list of the Top 10 go-to websites to help solve your PCR problem.
While the classic approach to molecular cloning – using restriction enzymes to excise a DNA fragment of interest – is as useful as ever, new techniques that make cloning faster, easier and more versatile are available. As a smart molecular biologist, you should be examining each of them to see whether or not adding them…
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