PCR, qPCR and qRT-PCR
What is PCR? – The Beginner’s Guide
If you need to copy, sequence, or quantify DNA, you need to know PCR. But what is PCR? Read our guide to the PCR process, and discover tips to help you avoid the most common PCR pitfalls.
Read More A Step-by-Step Guide to Designing qPCR Primers
qPCR primer design is a bit of science, a bit of magic, and a little bit of luck. Here’s the science to help you design the best primers for your experiments.
Read More 4 Easy Steps to Analyze Your qPCR Data Using Double Delta Ct Analysis
New to qPCR? Here’s a quick summary of one of the two most common analysis methods – double delta Ct analysis.
Read More 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!
Read More What Is a Cq (Ct) Value?
Don’t stay confused by Ct values! We’ll guide you through what they are, how to calculate them, and troubleshooting issues.
Read More How to Prevent False Results in Colony PCR
How to Prevent False Results in Colony PCR Colony PCR saves time and reduces costs by eliminating the need for plasmid purification. However, confounding results abound — but only if you fail to anticipate them. This article outlines the major perpetrators of false results and how to prevent them. For a more general overview of…
Read More 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…
Read More Site-Directed Mutagenesis Tips and Tricks
There are a few different ways of approaching site-directed mutagenesis. Here, I’ll give you a quick introduction to inverse PCR and why it’s useful, as well as going through a full protocol for SDM using modified primers!
Read More 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…
Read More 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…
Read More 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,…
Read More 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…
Read More 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…
Read More 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…
Read MoreDecisions, 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,…
Read More 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…
Read More 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…
Read More 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,…
Read MoreIntercalating 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…
Read More 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…
Read More 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…
Read MorePCR 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…
Read More 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…
Read More 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…
Read More 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…
Read MoreSmall 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…
Read More 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,…
Read More 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…
Read More 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…
Read More 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…
Read More 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…
Read More 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…
Read More 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,…
Read More How to Choose the Right Pipette Tips for your Experiment
The precision and accuracy of even the best calibrated pipette can be wiped out if you choose the wrong kind of tips. Depending on the experiment you are doing, the wrong kind of tips can also make your pipette a source of contamination, lead to waste of precious samples or reagents—or even cause you physical…
Read More 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…
Read More 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…
Read More 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…
Read More 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.…
Read More 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.…
Read More 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…
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