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PCR, qPCR and qRT-PCR

MIQE Guidelines for Digital PCR

MIQE what’s that? When writing dPCR materials and methods for a paper have you ever pondered what information you should include? This is where the MIQE guidelines will really help. Guidelines for minimum information required for publication of a digital PCR (dPCR) experiment were published by JF Huggett et al. in 2013. These were a…

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The A-Z of PCR Variants

The wide range of applications of PCR has led to an ever-growing list of variants of the technique. While some are optimizations to suit specific requirements and are very similar to basic PCR, others completely turn the technique on its head to formulate novel creative applications in various fields. This article lists some variants of…

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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…

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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…

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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…

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How Are Crimes Solved by PCR?

The Scene of the Crime The body of a woman is found behind an abandoned warehouse on the outskirts of town. Forensic experts carry out a technical examination of the scene and suggest strangulation as the cause of death. The absence of bloody footprints or weapons means there are no obvious leads to the killer.…

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Choosing the Correct Enzyme for PCR

A wide variety of enzymes are available for PCR and RT-PCR and the optimal choice depends on a range of factors specific to your experiment. Some of these factors will now be explored to help you to make the most suitable and cost effective choices when ordering. PCR Type and Other Factors to Consider First,…

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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…

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Just What Do All These Additives Do?

Every PCR battle is the same: Too little amplification of your target DNA versus too much amplification of off-target DNA. But you can win the PCR battle and amaze your co-workers by mastering the use of PCR additives. PCR additives usually work one of two ways: 1) By reducing secondary DNA structures and thus increasing…

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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…

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What is Digital PCR?

Digital PCR (dPCR) is a quantitative PCR method that provides a sensitive and reproducible way of measuring the amount of DNA or RNA present in a sample. This method is similar to qPCR in the reaction assembly components and amplification reaction, but differs in the way the sample target is measured.  Digital PCR is a…

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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…

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A Primer for Designing Degenerate Primers

For those of us who work with Mus musculus or Homo sapiens, to name a couple of species, a few clicks on UCSC Genome Bioinformatics Site or Ensembl gets you the full and precise DNA sequence for any annotated gene in the genome. This luxury is not in place for all species however; many of…

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How RNA-FISH Can Complement qPCR, and Vice Versa

In studies of RNA abundance and gene expression, no one technique can answer all of the questions that need to be asked. So it is necessary to use a variety of experimental methods in concert. Two RNA detection and measurement techniques that complement each other well for this purpose are RNA Fluorescence in situ hybridization…

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A primer on Probe-based SNP genotyping

Single Nucleotide polymorphisms (SNPs), colloquially pronounced ‘snips’, are the most common type of genetic variation in people. By definition, a SNP represents a single nucleotide variation at a specific location in the genome that is found in more than 1% in the population. For example, a SNP can replace the nucleotide cytosine (C) with an…

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How to scale up a PCR reaction

Scientific creativity and repetitive tasks do not go well together.  There is nothing worse than having to repeat the same task over and over again – I know somebody who quit science because of this and became a castle curator. So how do you minimise the amount of one of the most repetitive tasks –…

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A Quick Tour Around Probe-based Multiplexing qPCR

PCR has become the tool of choice for molecular diagnostics and is now a staple platform in any laboratory setting. The versatility of this method has led to a myriad of spin-off techniques, including probe-based quantitative PCR (qPCR). This method effectively combines PCR amplification and detection into a single step to measure the specific amount…

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qPCR Assay Design 101

Links to Resources mentioned in this webinar Real Time Design: real time qPCR probe design application from Biosearch Technologies Real time PCR probe animation video BHQ Dye Selection Chart Webinar Abstract Good probe and primer design is at the heart of any successful real-time qPCR assay. Watch this webinar to learn the principles of oligo…

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How to Screen for CpG Methylation by Methylation Specific PCR

In a recent article, I gave some tips about how to obtain good results with sequencing DNA after bisulfite conversion (it contains some tips that apply to the approach described in this article, too). Bisulfite sequencing is a very useful technique if you want to know the methylation status of every CpG in your genomic…

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Do Your Homework to Find Good Reference Genes

Comparing and measuring gene expression is certainly an integral part of research—gene expression patterns continue to show us how different cell networks are regulated, and point to new biological pathways and possible treatments for disease. But one crucial part of gene expression lies in making sure that differences in gene expression are due to gene…

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How to Calculate a DNA Primer Concentration

Calculations can be the bane of laboratory work. Fortunately, there are many easy methods to help you do the maths you need in the lab. Here, we tell you about the different ways to calculate primer concentration depending on the starting material. For all calculations, let’s assume we have 22 nmol of a DNA primer…

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How to Amplify Difficult PCR Substrates

During my postgraduate studies, I did literally one PCR reaction with a pre-optimised protocol on a not especially difficult template. So my karma came back with vengeance, when as a part of my first postdoc I had to amplify a template containing a 35 bp-long GC-rich stem-loop, which proved to be extremely difficult. This was…

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Mind Your P’s And Q’s: A Short Primer On Proofreading Polymerases

For applications such as site-directed mutagenesis, it is often recommended that you use a proofreading polymerase (also known as high-fidelity polymerases) to minimize the risk of introducing unintended point mutations.  But what is a proofreading polymerase?  What makes them different from other polymerases?  And when should you use them?  Read on to learn more… What…

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The Art of PCR Primer Design

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…

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Let’s Get Melting

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).…

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MIQE Guidelines: Do Your RT-qPCRs Make The Grade?

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…

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PCR: The Right Way to Decontaminate and Eliminate False Positives

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…

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PCR vs qPCR Sensitivity

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…

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Better Than Betaine: PCR Additives That Actually Work

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…

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Read This Before You Design Those qPCR Primers

qPCR is a technique used daily in most labs, but the first step, designing your qPCR primers, can be the biggest obstacle to even getting started. Without a good pair of primers, you can’t start asking the real questions and generating data.  And sometimes the effort involved in optimizing an assay for high efficiency and sensitivity…

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qPCR: Plexor vs Hydrolysis Probes

Hydrolysis probes, commonly also referred to as TaqmanTM is a very popular chemistry for real-time PCR. In this article I will compare hydrolysis probes with PlexorTM. But first, a quick overview of hydrolysis probes. Hydrolysis probes, an overview Hydrolysis probes are a popular detection chemistry for monitoring sequence-specific amplification in RTPCR. Just like with  SYBR…

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qPCR: Plexor and SYBR compared

In my last article I introduced you to the Plexor System. And from that we already know that while in reactions that user SYBR Green for detection, fluorescence increases with accumulation of PCR product, with Plexor the fluoresence decreases. Today I want to compare some other well-known features of SYBR Green chemistry and see how…

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Get the qPCR Fluorescence Low Down with Plexor

In real-time PCR, there are two primary ways to detect amplicons using fluorescent monitoring. One is intercalator-based dyes such as SYBR Green, and the other is probe-based techniques (hydrolysis or hybridization probes). All of these methods share a similar mechanism of measuring increasing fluorescence during amplification. But there is another completely different way to quantitatively…

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The Secrets of Thermophile Survival: Part I

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…

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The Taq behind PCR

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…

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Quantitative RT-PCR: One-step or Two-step RT?

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…

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The Invention of PCR

Few technical breakthroughs have changed the face of their field like the Polymerase Chain Reaction (PCR). Gene cloning, sequencing of complex genomes, DNA fingerprinting and DNA-based diagnostics are just some of the techniques that were either inefficient, crude or plain impossible before PCR. The technique has revolutionized biological research and biotechnology to such an extent…

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