The qRT-rtPCR Control You Should Be Doing, But Probably Aren’t

The qRT-rtPCR Control You Should Be Doing, But Probably Aren’t

Every man, woman, and dog is doing quantitative real time reverse transcriptase PCR (qRT-rtPCR) these days. It’s a great method to measure your favorite transcript’s expression levels. One of the big plusses (like the Swiss flag!) of quantitative PCR in general is its high sensitivity. In principle, it can detect and quantify one molecule of…

Oligo Purification Methods: How, Why and for What?

Oligo Purification Methods: How, Why and for What?

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…

PCR Pitfalls: The Devil is in the Details

PCR Pitfalls: The Devil is in the Details

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…

Divide and Conquer: How to Setup Your First Droplet Digital PCR Experiment

Divide and Conquer: How to Setup Your First Droplet Digital PCR Experiment

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…

Small Particles (Things) Matter!- Introducing Nanoparticle PCR

Small Particles (Things) Matter!- Introducing Nanoparticle PCR

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…

From Revolution to Evolution: Stem-loop Real-time PCR

From Revolution to Evolution: Stem-loop Real-time PCR

Kary Mullis invented polymerase chain reaction (PCR) in 1985 creating a revolution in molecular biology techniques. But it hasn’t stopped there. PCR has greatly evolved over the years. Today, we stand at a point, where we can clone micro RNAs (miRNAs) in real time! Due to miRNA size (about 18-21 nucleotides long) and varied expression levels,…

SPUD’s Your Bud When it Comes to Sensitive qPCR

SPUD’s Your Bud When it Comes to Sensitive qPCR

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….

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…

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…

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…

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…

Digital PCR or Quantitative Real-Time PCR: Which Method Is Best for Your Quantitative PCR Application?

Digital PCR or Quantitative Real-Time PCR: Which Method Is Best for Your Quantitative PCR Application?

So you’re designing a new experiment that requires PCR quantification. You used to have only one method to choose from, but now you have two – Quantitative Real-Time PCR (qPCR) and Digital PCR (dPCR). Which one is right for your application? Both methods have good quantification, sensitivity and specificity for most applications. They are compatible…

Western Blot, ELISA, SPR, Biosensor Assay or PCR: Which Technique Should I Use?
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Western Blot, ELISA, SPR, Biosensor Assay or PCR: Which Technique Should I Use?

Stimulation of cells/tissue with a given stimulus (e.g., a cytokine) is a common experimental setup in any cell biology lab. The cellular response to the external stimulus e.g., the activation/deactivation of intracellular signaling pathways and/or the secretion of proteins is often the research goal, and there are a number of different methods that you can use to analyze such…

Apps That Bring PCR to Your Mobile Device

Mobile devices such as iPads and smartphones are increasingly being used by laboratory researchers to aid them in their research. One example is the move towards electronic lab books. As a result, app developers have responded to this trend and are developing apps tailored to specific laboratory techniques including PCR and qPCR. The following are…

How to Train a Junior Colleague in the Basics of PCR

How to Train a Junior Colleague in the Basics of PCR

You’re a senior grad student or postdoc, and you’ve done more PCRs than you can count. A new student has joined your lab, and you’ve been charged with training them on PCR. You don’t want to lead him/her astray, but it’s hard to remember the parts that you struggled with in your early days. This…