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…
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