Qiagen - Bitesize Bio

Multiplex Ligation-dependent Probe Amplification (MLPA)

Multiplex ligation-dependent probe amplification (MLPA) is a molecular technique developed by MRC-Holland back in 2002. In a nutshell, MLPA is a sensitive technique that allows quantification of nucleic acid sequences, quickly and efficiently. It is performed in many laboratories worldwide, and can be applied to detect copy number changes (like deletions or duplications) of a…
Using CRISPR/Cas9 to detect sequences on single DNA molecules with high-speed AFM

Join Dr. Jason Reed as he describes a novel method by which endonuclease-inhibited Cas9 can be employed as a programmable biomarker in high-speed atomic force microscopy (HS-AFM) imaging. In this webinar, you will learn: How CRISPR/Cas9 can be used to “flag” alterations and mutations in DNA, rather than cut it How pairing atomic force microscopy…
A Start to Finish Guide to Target Gene Validation Using Quantitative RT-PCR

A Start to Finish Guide to Target Gene Validation Using Quantitative RT-PCR Speaker Matthew Mule In this tutorial, you will find: While next generation sequencing enables researchers to unveil expression levels of the entire genome, qRT-PCR remains the gold standard for measuring transcript levels of individual genes for functional studies and for the purposes of…
The Use of qPCR to Validate Epigenetic Enrichment of Pathogen DNA from Complex Samples and Human DNA from Stool

qPCR is one of the most specific and sensitive tools in molecular biology, allowing the quantification of target DNA molecules present at less than 1 in 106. Next Generation Sequencing (NGS) has similar potential. However, the presence of large amounts of non-target DNA in most clinical or environmental samples precludes easy and inexpensive analysis of…
Common challenges and solutions in microbiome analysis workflows

Elucidating meaningful, unbiased microbial community profiles from complex microbiome samples is challenging. In this webinar, you will learn: – the sources of bias throughout the microbiome analysis workflow – practical solutions for troubleshooting your techniques – new technologies to achieve the most representative and unbiased microbiome profiles Join Dr. Sven Reister as he guides you…
Immuno-PCR: A Highly Sensitive Method of Immunodetection

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…
Long Adapter Single Stranded Oligonucleotide (LASSO) Probes for Massively Multiplexed Cloning of Kilobase-Sized Genome Regions

In this webinar, you will learn how to solve a major problem in creating expression libraries from genome sequences for downstream analyses. Specifically, you will learn: The difference and benefits of LASSO cloning over NGS How LASSO cloning allows for the multiplex cloning of large open reading frames (ORFs) of bacterial, human, and human-microbiome genomes…
Small Differences that Matter: Detecting Microsatellite Polymorphisms

If you have any training in genetics, chances are that during the course of your education you ran into those funny little sequences called microsatellites. These are repeated tandem motifs 1-6 nucleotides long, scattered all over our genomes. These used to be called “junk DNA,” because researchers thought that the repeats served no purpose. Nowadays,…
Primer Validation Was a No-Go – Now What?

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…
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…
Quantifying Allele-Specific Gene Expression Using PCR-Based Methods

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…
Decisions, Decisions: How to Choose the Best qPCR Probe for Your Experiment

Before we go any further, we have to get some things straightened out: RT-PCR versus qPCR versus RT-qPCR. Sooo confusing, amirite?? They all refer to specific molecular biology assays, but the names are unfortunately used interchangeably, which can be awfully confusing for just about anyone. So without further ado: RT-PCR is short for reverse-transcriptase PCR,…
How to Survive a Difficult PCR

I am sure many of you have been there. Everything is going smoothly, and your project seems to be working out perfectly. And then there is this one PCR. For some reason, it just won’t work. It is a black dot on your record. Even though I have a scientific mind, I have to be…
Amplify Your PCR Success with the Right PCR Instrument!

Nowadays, almost every biology lab has a PCR instrument — from portable, battery-operated machines, to “PCR-by-water baths”, do-it-yourself PCR, or familiar vendor packages, including those with real-time quantification or droplet digital capabilities, DNA amplification depends on a robust thermal cycler. If you are looking to upgrade, add to, or even replace the lab’s current PCR…
Microsoft Excel Can Help You Set Up Multi-Well Plates

When you are a newbie to qPCR or qRT-PCR, it is quite common to write everything down in your lab notebook and then tediously mark off reagents or samples as you add them. People typically start with a master mix for items you add to multiple samples such as (Mg2+, dNTPs, 10X PCR buffer, additives,…
Intercalating Dyes or Fluorescent Probes For RT-qPCR?

The unique feature of real-time quantitative polymerase chain reaction (RT-qPCR) is that it associates the amplification of your target gene with a fluorescent signal in a quantifiable manner. Presently, there are numerous fluorescent tool kits/methods to consider when designing your RT-qPCR experiment. However, the two major categories to choose from are fluorescent intercalating dyes and…
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…
The Inhibitors Haunting Your PCR

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 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…
qPCR and RNAseq: The Battle of the Strands!

In this webinar, you will learn how qPCR can be used in combination with new technologies, like RNAseq, and how this will help you in your research. The main aspects to be covered in this webinar include: When you should choose RNA-seq over qPCR alone for expression analysis Why and how you should validate RNAseq…
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…
How to Choose the Correct Reverse Transcription Method

Quantitative Reverse transcription PCR (RT-qPCR) is frequently used in the lab to detect and quantify RNA expression in a sample. The first step of the assay is to convert the labile RNA to its complementary DNA (cDNA) counterpart through reverse transcription (RT). In fact, RT is the first step in a variety of molecular biology…
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…
An Introduction to MassTag PCR

New ways to perform PCR emerge all the time. This speaks for the speed of technological advances, and reflects the ongoing need to keep up with fast-moving research. We all know that PCR’s main purpose is to amplify a stretch of nucleic acids based on sequence-specific primers. Nowadays, a wide range of PCR techniques exist,…
Reverse Transcription: The Most Common Pitfalls!

Good quality starting material is king for reverse transcription! Obtaining reliable results in any experiment requires good preparation. We often take reverse transcription for granted, and we don’t always consider that our qPCR might be performing poorly because of problems in that step. Since it’s quite often the reverse transcription reaction itself that causes fuss…
Faster PCR Optimization

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…
Multiplex PCR Technology: What Is It All About?

Join Dr. Karen O’Hanlon Cohrt for a practical tour of multiplex PCR technology, where you will learn the following and much more: Principle behind multiplex PCR technology Popular applications of this technology How to set up this reaction Advantages and disadvantages Multiplex PCR Can Benefit Your Research Dr O’Hanlon Cohrt will discuss the history of…
The Obligate qPCR Standard Curve

At first sight, real-time PCR looks like a very simple technique—very straightforward. Also, when it’s optimized, real-time PCR leads to interesting results. However, to obtain consistent and accurate results reflecting the reality, good controls are crucial for SYBR qPCR. One of these controls is the qPCR standard curve to check for the efficiency of your primers. Efficient Primers…
DNA Shuffling Like a Pro

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…
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,…
The Real-Time PCR Digest

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…
Methods for Relative Quantification of qPCR Data. Yes, There is More Than One.

As all of you probably know, methods for calculating relative gene expression from qPCR data include: a) double delta Ct (ΔΔCt) and b) that one other method. Chances are you’ve probably gotten beyond the ΔΔCt method, but you should be prepared in case you face primer sets of different amplification efficiencies. Both methods require the…
Decoding Cancer: Practical Advice for Working with Cellular Heterogeneity

In this webinar, you will learn about the process for genotyping cells—particularly tumor cells—and practical advice for analyzing the resulting data. The main points that we will cover are: Practical advice and methods for sample collection and preparation for your genotyping experiments An overview of the available data analysis methods for your genotyping results Examples…
The Essential PCR Troubleshooting Checklist

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.…
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.…
All about miRNAs: Practical Tips, Advice, and Applications

In this webinar you will learn the tips and tricks necessary for you to successfully work with miRNAs, and how you can use them to further your research. The main points we will cover are: Tips and practical advice to help you work with miRNAs, including isolation and delivery The biology and mechanism of miRNAs and…
PCR Problems? Try an Additive

You’ve tried all the usual stuff, and checked the primer sequences twice, but still can’t get that PCR fragment amplified. It’s time to enter the strange world of PCR additives. Over the years a variety of additives have been shown to enhance PCR reactions in certain situations. Here is a summary of some of the…
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…
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…
Entering the Digital Age for Quantitative PCR Analysis: Digital PCR

Digital PCR (dPCR) is a next generation qPCR that you might just need for quantitation and comparison of minute genetic copy differences.
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 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…
Touchdown PCR: A Primer and Some Tips

When I first heard of touchdown PCR, I thought of a landing aircraft, which, as it turns out is not a bad way to think about it. But despite it’s amenability to analogies and dreadful puns (see title), touch-down PCR (TD-PCR), a very useful technique for improving PCR amplification specificity, is trickier that it might…
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…
2015 Staff Picks: PCR & Real-time PCR

To wrap up 2015, we’re highlighting our favorite articles from the year. These articles were handpicked by our editorial staff here at Bitesize Bio. So, sit back with your cup of coffee (or beverage of choice) and enjoy these fantastic articles from 2015. First up, we have the category of PCR & Real-time PCR. Jen…
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…
Are You Ready for Your First qPCR?

Hope you had a heavy breakfast, because the first qPCR is going to take time. Did you remember to fill your coffee cup? There are a lot of intricacies in qPCR that will need your neural networks to be brisk. How qPCR differs from traditional PCR Unlike traditional PCR, qPCR measures the amplification of DNA…
4 Easy Steps to Analyze Your qPCR Data Using Double Delta Ct Analysis

You are at the airport burning away time with a report due tomorrow morning for your professor. You have your data. Why not take advantage of the time and calculate the expression fold change for the gene you have tested in the lab? It’s easy – I’ll show you how. What you need for analysis…
Life of the Party: Viability PCR (vPCR)

Viability PCR (vPCR) is a big step forward in PCR technology. Through the use of a simple pre-treatment of the sample(s) of interest using specific intercalating reagents, it is possible to neutralize the DNA of dead cells. As a result, only DNA from live cells will be amplified by PCR. Through the vPCR, it’s possible to…
Smartphone PCR Apps for PCR-On-The-Go

For the busy scientist, a Smartphone is more than just a Facebook and Instagram viewer. In the past few years, apps have been developed that can also allow you to use your phone or tablet to design PCR and qPCR experiments on the go.
Troublesome Site-Directed Mutagenesis: Troubleshooting Your Experiment for Stubborn Mutations

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!
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…
How to Quantify Integrated HIV Genomes Using Alu-gag PCR

Alu sequences are repetitive DNA sequences that are widely dispersed within the human genome. These “junk DNAs” are not as useless as one might think. An interesting method to use them is to quantify the number of integrated Human Immunodeficiency Virus (HIV) genome copies using Alu-PCR.
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…
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…
Get that Genotyping PCR to Work EVERY TIME

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…
qPCR Tips: Workflow, Applications and Troubleshooting

In this webinar, you’ll get: Practical advice for sample preparation, qPCR setup and result analyses Guidance on choosing the correct fluorescent labeling system for your qPCR Tips to troubleshoot the most commonly encountered issues in qPCR Join Dr. Karen O’Hanlon Cohrt for a practical tour of the process, applications and practice of qPCR, from start…
Single-cell Genomics: An Overview and Its Application in Investigating Complex Biological Samples

In this webinar you will learn: The science behind single-cell genomics and its applications How single-cell genomics applies to your research through an example study. In this study, single-cell genomics was used to study single cells from microbes in the environment. Practical advice and tips to help you perform single-cell genomics experiments and analysis of the data…
Next Generation Sequencing Library Preparation: Concepts, Tips and Tricks

In this webinar, you will learn: The core steps in preparing DNA/RNA NGS libraries An overview of the major strategies for generating NGS libraries Tips and tricks to generate high quality NGS libraries for consistent sequencing results The widespread availability NGS has caused a revolution in genetics and molecular biology research. While several NGS platforms…
Next-Generation Sequencing Overview: Step By Step Guide to NGS Workflow

In this webinar you will learn: Pre-analytical considerations – how different sample types can cause issues in downstream processing. Sample Preparation for the most common applications (Genomes, Exomes, Targeted Panels) – how choosing the right library construction method is critical to success. There is a big world out there – the many other sample preparation…
Critical Success Factors for Sample Collection, Storage and Retrieval

In this webinar you will learn: How to maintain specimen integrity when storing tissues, blood and cells. How to store and retrieve specimens from a biobank. Special considerations for preparing formalin-fixed paraffin-embedded (FFPE) tissues for storage. Abstract: Biobanks, or biorepositories, store biological samples that are critical for research in the biomedical fields. Samples from biobanks…
Liquid Biopsy Sample Handling

In this webinar you will learn: How to sensitively, specifically and rapidly analyze circulating free nucleic acids (cfDNA), circulating tumor cells (CTCs) or exosomes from a blood sample using liquid biopsy About miRNA signatures and how they have the potential to serve as biomarkers for disease pathogenesis, prognosis, and response to treatment. How to overcome…
What Is a Cq (Ct) Value?

Real-time PCR (often called qPCR) is usually conducted to quantify the absolute amount of a target sequence or to compare relative amounts of a target sequence between samples. This technique monitors amplification of the target in real-time via a target-specific fluorescent signal emitted during amplification. Despite the fact that real-time PCR fluorescent dyes and probes should be sequence-specific,…
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 still…
Get To Know Your DNA Polymerases

While most may think standard Taq is the backbone of PCR, there are many other DNA polymerase options out there. The polymerase you use has a significant impact on the efficacy of your PCR, specifically on the product yield, the purity of the product and the faithfulness with which the starting product is transcribed. Sometimes,…
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…
PCR on a shoestring: Build Your Own PCR Machine

Maybe you’re a teacher hoping to do something different, maybe you’re a student trying to drag out their funding, or maybe you just want to build something really cool. Did you know that PCR used to be done by hand? Imagine three water baths with thermometers, a timer going off every few minutes, and a…
Techniques for Isolating Circulating Biomarkers

In this webinar you will learn: Which biomarker you want to isolate Which clinical biofluid or specimen is a good biomarker source A basic understanding of small non-coding RNAs, exosome, and circulating tumor cell biology The challenges associated with isolating these biomarkers Various technologies to overcome these challenges Abstract: To gain a better molecular understanding…
10 Handy Tips to Help Keep Your PCRs Contamination Free

PCR contamination can be your worst nightmare if you are not able to figure out the source of the bands in your negative control. Initially, when I had to setup PCRs, contamination was my main problem and it remained a mystery for a while, as no matter how many times I repeated it with a…
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

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…
Free PCR for 5 Years (or How to Make your Own Taq and Pfu Polymerase)

Labs across the world spend a great deal of money on commercially manufactured Taq polymerase for PCR every year. But you don’t have to: Many labs choose to save money by making their own Taq (and Pfu for that matter) for routine applications. This post is not intended to provide a step-by-step protocol for making…
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

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…
Clean Up Your Act! How To Clean Up PCR Contamination

You carefully set up your PCR, excitedly waited for it to finish, ran your gel, and waited for the big reveal. But instead of seeing what you hoped (a nice clean gel), you see a big fat mess—extra bands and, most disturbingly, bands in your negative control. So, what are you to do? How do…
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…
Overhang PCR

Have you ever “accidently” forgotten to add the Kozack consensus sequence to the start of a coding genes? Or forgotten to include the stop codon? Did you clone something, then realize you wanted to tag it with something? Or you just want to add restriction enzymes to your PCR product to make it easier to…
An Essential Toolbox for qPCR Users

Today, PCR is as common a feature to the lab as pipettes and beakers. The majority of us regularly need to amplify our DNA or RNA samples, sometimes for an ‘everyday’ PCR run just to check if our primers actually work, or in a quantitative (q)PCR run, where we might be comparing the levels of…
Top Ten websites to help you with your PCR experiments

Whether you are embarking on a PhD in molecular biology, trying to amplify a new gene, analyzing gene expression by qPCR, or trying to solve a PCR problem, you will probably resort to a Google search at some point or another. How do you sort out the > 20 million hits you get when you…
Taking the Pain, Challenge and Rage out of PCR: The BitesizeBio Guide to PCR

Polymerase Chain Reaction, better known simply as PCR, has come a long way in the past 30 years. For those of you old enough to remember the not so ‘good old days’ when PCR was manually performed using a series of water baths and overlaying oil onto our precious samples (yes, seriously!), I think most…
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

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…
Thermal Asymmetric Interlaced PCR (TAIL-PCR)

What do bunnies, coins and PCR have in common? They all have tails! (ha ha!) Thermal asymmetric interlaced PCR or TAIL-PCR is used to sequence and analyse unknown DNA fragments that are adjacent to known sequences. Think of it as being rather like networking. You know you want to get to know someone so you…
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

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,…
Mischief in the Mastermix: Bothersome Bubbles

It happens to the best of us. You’re minding your own business and suddenly out of nowhere your mastermix is a bubble bath and your primers are enjoying a froth party. Let’s talk about how to deal with these foamy fiends! When you’re making up your mastermix, you could have a variety of ingredients going…
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…
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…
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

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

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…
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.…
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,…
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…
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…
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…
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…
The Future of PCR

After 30 years, what’s next for PCR? In the 80s, saying you were doing “PCR” was enough for everybody to know what kind of experiment you were performing. Today, the “PCR family” has gained so many new members that it can be confusing to decipher which type of PCR you’re using. Developed in 1983 by…
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…
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…
What is PCR? – The Beginner’s Guide

PCR is THE technique of modern molecular biology labs. If you need to copy, sequence or quantify DNA , you need to know PCR. In short, PCR (polymerase chain reaction) is a biochemical technique that uses thermocycling and enzymes to quickly and reliably copy DNA, and it was invented in a flash of inspiration by a…
8 Essential Papers and Reference Guides for Quantitative PCR (qPCR)

It can be daunting to learn about a new topic when you look at the literature and realize that thousands of papers have been published on the topic. This is especially true for techniques that have transformed scientific research. Quantitative PCR (qPCR) also known as Real-Time PCR (RT-PCR) has revolutionized the study of gene expression…
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 –…
A Step-by-Step Guide to Designing qPCR Primers

Primer design is a critical step when setting up your qPCR or reverse transcription-qPCR assay (RT-qPCR). qPCR primers that anneal poorly or to more than one sequence during amplification can significantly impact the quality and reliability of your results. Also, if you are performing a one-step RT-qPCR, the reverse transcriptase will use the reverse primer…
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…
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…
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…
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…
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…
Eliminate PCR Amplicon (but not real-time PCR?) Carry-Over With UNG

Using UNG is a great trick for PCR amplicon decontamination, but is it any good for rtPCR?
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…
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).…
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…
So You Think You Know PCR?

Well, after much anticipation, we finally got the Bitesize Bio seminars off the ground with a rip-roaring presentation from SYBR Green I and Lightcycler inventor, Carl Wittwer. Carl’s talk took us from the early days of rapid cycle PCR to the development of the Lightcycler through to the diversification of real time PCR into different…
Important Considerations for Determining qPCR Efficiency

One of the very first things you need to do when getting set up for quantitative PCR (qPCR) is to determine the efficiency of the assay because knowing the assay efficiency is critical to accurate data interpretation. And you have to do this every time you design and purchase a new primer pair. Ideally, the…
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…
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…
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…
Read This Before You Design Those qPCR Primers

qPCR is a technique used daily in most labs, but the first step, designing your 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 can be…
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…
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…
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…
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…
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…
Fast, Accurate and Green PCR

Attending the 2009 American Society of Microbiology meeting this week in Philadelphia, I decided to take my own advice and visit the exhibits to see whether any of the exhibiting companies had any really innovative and unique products on display. I focused mainly on products I might use in my own lab, such as PCR…
A New Use for Acupuncture in qPCR

What do you get when you cross eastern medicine with molecular biology? How about a novel way to isolate mRNA from living cells using acupuncture? That’s just one of many projects going on in Dr. Karl Hasenstein’s lab at the University of Louisiana, Lafayette. Original research performed by Dr. Hasenstein’s lab and presented at the CHI…
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…
The PCR song

An extremely cheesy but fun promo from BioRad
The Best Polymerases of 2008

Yawn…. The awards season is upon us once again. Overpaid, under-worked and over-ego’d celebrities get together to slap each other’s backs and tell each other how great they are. But little do they know where the real party in town is. The 2008 Thermostable Polymerase Awards (the THEPA’s) are underway and you have a front…
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…
My favorite PCR polymerase

What do you get when you cross a Pyrococcus DNA polymerase with a dsDNA binding domain? …a highly processive, high fidelity, lighting fast PCR work-horseThose Finnish wizards at Finnzymes have made that work-horse a reality with their Phusion polymerase. I had been meaning to try out this enzyme for quite a while, but never got…