No More White Elephants! – Consider this Before Buying a Real-time PCR Cycler

No More White Elephants! – Consider this Before Buying a Real-time PCR Cycler

Does your lab have a closet full of white elephants; once expensive instruments that are no longer fit for purpose, or have broken down? In many cases, all of that wasted money and resource could have been saved if the buyers had made smart choices about matching the instrument more closely to their needs. A…

Do You Know Where You Are? Find Out with Genome Walking

Do You Know Where You Are? Find Out with Genome Walking

Do you work with plants? Are they genetically engineered? Do you know where and how? If not, you could experience problems. After all you do not want your transgenic gene cassette to disrupt genes that would affect your phenotype of interest. In this article I will tell you about Agrobacterium-mediated transformation – a widely used…

Long-Range PCR:  It’s About Choosing the Right Enzyme

Long-Range PCR: It’s About Choosing the Right Enzyme

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…

10 Tips for Consistent qPCR

10 Tips for Consistent qPCR

Quantitative PCR (qPCR) uses fluorescent dyes or probes to visualize the amplification of specific DNA sequences as it happens (i.e. in real time). The dyes or probes fluoresce when they bind to newly amplified DNA, and the amount of fluorescence emitted is proportional to the amount of DNA (or mRNA) present in the original sample. By detecting newly synthesized DNA…

Photonic PCR: When Lightening Strikes Your DNA

Photonic PCR: When Lightening Strikes Your DNA

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…

Time to Instigate Nested PCR

Time to Instigate Nested PCR

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…

How Thermophilic Bacteria Survive, Part II: DNA

How Thermophilic Bacteria Survive, Part II: DNA

In part I, I answered the question, “How do proteins in thermophiles survive under high temperatures?” In this part, I’ll look look at how nucleic acids survive -thrive, even- in conditions that are too hot for most of us, but ideal for a number of organisms, including the one that gave us Taq polymerase and…

Control your error! How to minimize pipetting error and get low SDs in qRT-PCR

Control your error! How to minimize pipetting error and get low SDs in qRT-PCR

Variability is the Achilles’ heel of research. It can often confound our results and lead us astray searching for solutions. There are two kinds of variability, the first is biological variability. This represents the stochastic nature of the sample you are working with and the inherent differences between samples from the same conditions. There is…

When PCR Gets RACE-y: From Unknown mRNA Segments to Sequenced cDNA

When PCR Gets RACE-y: From Unknown mRNA Segments to Sequenced cDNA

Normally you need two primers to amplify your segment of interest – one for the 3′ end of your segment of interest and one for your 5′ end. But if you don’t know the sequence of the regions you’re hoping to amplify this can be a problem! Rapid Amplification of cDNA Ends (RACE) is a…

Designing Luck: 8 Basic Concepts for Designing Primers for a Standard PCR

Designing Luck: 8 Basic Concepts for Designing Primers for a Standard PCR

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…

To boil? Or be boiled?  Saving Time With Colony PCR

To boil? Or be boiled? Saving Time With Colony PCR

Applying molecular techniques to unicellar organisms leads to many questions… Did my electroporation work? Is my vector inside my competent cell? Do I have contamination in my liquid culture? Is this the correct bacterial strain the neighboring lab promised me it is? Did the guy from the other side of the world send me the…

Detecting Signal in qPCR: From DNA Binding Dyes to BHQ Probes
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Detecting Signal in qPCR: From DNA Binding Dyes to BHQ Probes

A Brief History of Detecting Amplicons The Old Days of Ethidium Bromide In the early 1990s, quantitative (q) PCR was in its infancy, and despite PCR itself already being around for 10 years, there were no easy ways of precisely quantifying the amount of DNA that was amplified in a PCR reaction. In those days PCR…

Keeping On Top of Housekeeping Genes

Keeping On Top of Housekeeping Genes

Want to measure how much mRNA you have in a particular sample? Easy! Make some cDNA, add some fluorescent DNA-intercalating dye, pop it into a quantitative real-time PCR (qRT-PCR) machine and Bob’s your uncle! You have your result! Easy right…? Not so fast. As with any scientific assay, qRT-PCR requires some optimization. First, you need…

How to Be Greener – The Environmentally Friendly Guide to PCR

How to Be Greener – The Environmentally Friendly Guide to PCR

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…

Modify Your Oligos, Modify Your Experiments
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Modify Your Oligos, Modify Your Experiments

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…

Loop-mediated Isothermal Amplification

Loop-mediated Isothermal Amplification

Loop-mediated Isothermal amplification (LAMP), is an emerging technology that allows DNA amplification at a constant temperature. The key to this principle is the use of a DNA polymerase that possesses strand displacement activity. As a result of this property there is no need for heat denaturation of double stranded DNA in order to allow primer…

Finding Nemo: Understanding Single Cell Isolation and PCR Amplification

Finding Nemo: Understanding Single Cell Isolation and PCR Amplification

Every protocol for single cell PCR can be broken down into two steps. In the first step, the cells are isolated by micromanipulation, laser capture microdissection, flow cytometry, or by direct micropipetting. Next, the genetic material is processed by PCR to amplify your sequence of interest. Here, we’ll go through the different options for isolating…

qPCR: RNA Quality and Why It Matters

qPCR: RNA Quality and Why It Matters

Gene expression analysis plays a pivotal role in a wide range of studies, including biomedical analysis and diagnostics. Of all the methods available for gene expression analysis, quantitative real-time PCR (qRT-PCR) is the most rapid, sensitive, and accurate to measure mRNA, and its use in clinical diagnostics is rising steadily. RNA quality entails both purity and…

Mediator Probe PCR

Mediator Probe PCR

Want a PCR method that gives you high sensitivity and is cost-effective? Then you might want to try Mediator Probe PCR.  This method gives you sensitivity and limit of detection comparable to that of dual-labeled hydrolysis probes without the high cost. We all know how expensive small batch probe synthesis orders can be, especially when…

Understand and Troubleshoot PCR with The BitesizeBio Guide to PCR

Understand and Troubleshoot PCR with The BitesizeBio Guide to PCR

Over the years, I’ve had my fair share of ups and downs in the lab. The latter quite often centering on a failed or plainly weird PCR experiment. As I’ve gone on and become ever more fastidious about my lab practices I’ve realized that the majority of these little calamities were perfectly avoidable. In my…

Polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA)

Polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA)

As researchers, we are constantly on the lookout for new and improved ways to analyze, detect and quantify our favorite protein or gene. Luckily, we don’t always need to reinvent the wheel! PCR-ELISA is a good example of where two commonly used techniques have been merged together to create a very powerful analytical tool. What…

The History of PCR

The History of PCR

As with some of the greatest discoveries in science, from penicillin to microwave ovens and play-doh, PCR was discovered serendipitously. Thanks to the work of many scientists, including Watson and Crick, Kornberg, Khorana, Klenow, Kleppe (so many K’s…) and Sanger, all the main ingredients for PCR had been described by 1980. Like butter, flour, eggs,…

The Devil is in the Details: How to Setup a PCR Laboratory

The Devil is in the Details: How to Setup a PCR Laboratory

There is a right way and a wrong way to set up a PCR laboratory. Because of PCR’s tremendous ability to amplify small quantities of DNA/RNA template, even the smallest of template contamination can become a huge problem in PCR. However, contamination does not have to be a problem in your laboratory. Read below to…

Do-it-Yourself PCR

Do-it-Yourself PCR

Currently Open Source principles are offering interesting tools for doing molecular biology at an incredibly low cost. One interesting example is OpenPCR (www.openPCR.org) a project developed in order to ensure that the basic technology to perform PCR is affordably and openly available to all. In the past one of the main barriers for introducing PCR technology…

The different Phases of PCR and Why They Are Important

The different Phases of PCR and Why They Are Important

PCR (Polymerase Chain Reaction) is a biochemical technique developed by Kary Mullis in 1983 that is used to create large quantities of a sequence of DNA. Since this method of mass-producing DNA was first introduced, it has become significantly less labour intensive, more economical, and more routine. The technique relies on a few key players…