PCR, qPCR and qRT-PCR
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.
Read MoreControl 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…
Read MorePCR 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…
Read MoreWhen 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…
Read MoreNo 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…
Read MoreHow 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.
Read MoreEntering 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.
Read More10 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…
Read MoreDo 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…
Read MoreLong-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…
Read MorePhotonic 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…
Read MoreGet 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…
Read MoreTime 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…
Read MoreDesigning 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…
Read MoreGet To Know Your DNA Polymerases
While most may think standard Taq is the backbone of PCR, many other DNA polymerase options exist. The polymerase you use significantly impacts 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, these matter less, and quick and…
Read MoreTo 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…
Read MoreDetecting 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…
Read More10 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…
Read MoreKeeping 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…
Read MoreHow 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…
Read MoreFree 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 Taq polymerase. Find out how to save your lab some money.
Read MoreModify 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…
Read MoreLoop-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…
Read MoreFinding 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…
Read MoreClean Up Your Act! How To Clean Up PCR Contamination
The biggest source of PCR contamination is aerosolized PCR products. How do you fix PCR contamination and avoid it in the future?
Read MoreqPCR: 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…
Read MoreAn 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…
Read MoreTop 10 websites to help you with your PCR experiments
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.
Read MoreTaking 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…
Read MoreMediator 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…
Read MoreUnderstand 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…
Read MoreThermal 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…
Read MorePolymerase 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…
Read MoreThe 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,…
Read MoreMischief 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…
Read MoreMIQE 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…
Read MoreThe 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…
Read MoreThe 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…
Read MoreDo-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…
Read MoreThe 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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