Lyophilization is a process designed to remove water from a sample or product through a controlled freezing and vacuum application. The method leverages the triple point of water, where solid, liquid, and gas phases coexist under specific temperature and pressure conditions. The result is a room temperature stable product that is much lighter than the original sample or product.
Continue reading “An Introduction to Lyophilization: Process, Benefits & Possibilities”Imagine a scenario—you’re studying the developmental biology of a species of squid. The squid don’t reproduce in captivity, so females carrying fertilized eggs are collected from the wild and rehomed in your lab’s aquariums. You’ve monitored all the normal aquarium conditions—pH, temperature, salinity—ensuring the animal’s new home mimics its natural environment.
But then, for no reason apparent to you, the clutch of eggs doesn’t develop and doesn’t hatch, derailing your research program until next year when you can collect more adult squid from the wild. What went wrong?
As the SARS‐CoV‐2 pandemic continues to rage across the United States and around the globe, the demand for COVID‐19 testing is increasing. The vast majority of the COVID-19 assays use RT‐qPCR to detect the viral RNA in patient samples such as nasopharyngeal swabs, which are collected and stored in viral or universal transport media (VTM/UTM). The general workflow for these COVID‐19 assays can be broken down as follows:
While many companies who manufacture the products that are used in these steps have been able to adapt and significantly increase their production capacities, there are still gaps between the supply of these products and the global test demand. Both the sample collection and storage step and the RNA extraction/purification step have a tendency to bottleneck and experience supply constraints. One way to address these bottlenecks and expand production capacity for these in‐demand products is to evaluate the viability of skipping a step in the workflow, without hindering the ability to detect viral RNA from samples.
Continue reading “XpressAmp™ Direct Amplification: Simplified and Accelerated Time to qPCR Results”
The three winners of the 2019 Real-Time PCR Grants have been hard at work in the six months since receiving their grants. Each winner was eligible to receive up to $10,000 in free PCR reagents as well as the opportunity to collaborate with our knowledgeable technical service and training teams.
Abbeah Navasca is a plant pathology researcher with the Tagum Agricultural Development Company, Inc. (TADECO*, Philippines). She is developing treatments for viral infections that affect one of Philippines’ largest and most valuable agricultural exports: bananas. As a result of the qPCR grant, she and two of her colleagues were able to participate in sample preparation and analysis workshops with Promega Technical Services experts in Singapore. During her visit, the team worked through strategies for plant sample preparation and amplified those samples with the GoTaq® 1-Step RT-qPCR System. We had a chance to ask her more before she headed back to her lab.
Continue reading “Researching the Researcher: Abbeah Navasca, 2019 Real-Time PCR Grant Winner”
2019 Real-Time PCR Grant
The three 2019 Real-Time PCR Grant Winners have been hard at work in the six months since winning their grants. Each winner was eligible to receive up to $10,000 in free PCR reagents as well as the opportunity to collaborate with our knowledgeable technical service and training teams.
One of the 2019 winners, Alberto Biscontin (University of Padova, Italy), performs research in the fields of Neurogenetics and Chronobiology. He is looking to shed greater light on the circadian rhythms of the Antarctic krill. Alberto published his most recent analysis in Nature and GoTaq® qPCR Master Mix helped him validate expression of genes for his study.
His qPCR data showed support for internal mechanisms that not only support daily living but also clarified the overwintering process of the krill. Now that Alberto has sized up some zooplankton, we asked him to share a little more about himself and his research:
Q: How long have you been a researcher?
A: I have been a researcher since 2012.
Q: How did you decide to research Antarctic krill?
A: In 2013, I had the opportunity to join the international Antarctic research program PolarTime. [It] brought together eight research groups with different scientific expertise to study seasonal and daily rhythms in the Antarctic krill Euphausia superba.
Q: When you are not busy at the bench, what do you like to do?
A: Traveling. I love strolling through open-air markets.
Q: Are there any tips or tricks you have learned that make your job easier?
A: You can easily switch from a classic RT-PCR protocol to a cheaper and faster One-step protocol using the same primers and temperatures.
Q: What comes next?
A: I would like to characterize the clock machinery of other polar organisms to understand whether high latitude clocks have developed similar strategies to cope with [the] polar environment. Moreover, a better understanding of marine circadian clocks could help to shed light on the evolution of the animal circadian machinery.
You can find Alberto’s most recent publication in Nature Scientific Reports. The 2020 Real-Time PCR Grant will be coming soon. For more information on the 2019 winners and information on the 2023 Grant, visit the Real-Time Grant web page. Be sure to follow us on social media for the most up-to-date information regarding the 2020 Grant, including application deadlines and winner notifications!
We’re committed to supporting scientists who are using molecular biology to make a difference. Learn more about our qPCR Grant program.
PCR amplification with a proofreading polymerase, like Pfu DNA polymerase, will leave you with a blunt end. However, another thermostable DNA polymerase, like Taq DNA Polymerase, adds a single nucleotide base to the 3’ end of the DNA fragment, usually an adenine, creating an “A” overhang. This “A” overhang can create difficulties when cloning the fragment is your end goal. You might consider creating a blunt end with Klenow or adding restriction sites to the ends of your PCR fragment by designing them in your primers. But why go through all those extra steps, when that “A” overhang allows efficient cloning of these fragments into T-Vectors such as the pGEM®-T Vectors? Fewer steps? Who can argue with that?
Continue reading “Cloning Blunt-Ended DNA Fragments is Hard: pGEM®-T Vectors Can Make It Easier.”
For those of us well versed in traditional, end-point PCR, wrapping our minds and methods around real-time or quantitative (qPCR) can be challenging. Here at Promega Connections, we are beginning a series of blogs designed to explain how qPCR works, things to consider when setting up and performing qPCR experiments, and what to look for in your results.
First, to get our bearings, let’s contrast traditional end-point PCR with qPCR.
| End-Point PCR | qPCR |
| Visualizes by agarose gel the amplified product AFTER it is produced (the end-point) | Visualizes amplification as it happens (in real time) via a detection instrument |
| Does not precisely measure the starting DNA or RNA | Measures how many copies of DNA or RNA you started with (quantitative = qPCR) |
| Less expensive; no special instruments required | More expensive; requires special instrumentation |
| Basic molecular biology technique | Requires slightly more technical prowess |
Quantitative PCR (qPCR) can be used to answer the same experimental questions as traditional end-point PCR: Detecting polymorphisms in DNA, amplifying low-abundance sequences for cloning or analysis, pathogen detection and others. However, the ability to observe amplification in real-time and detect the number of copies in the starting material can quantitate gene expression, measure DNA damage, and quantitate viral load in a sample and other applications.
Anytime that you are performing a reaction where something is copied over and over in an exponential fashion, contaminants are just as likely to be copied as the desired input. Quantitative PCR is subject to the same contamination concerns as end-point PCR, but those concerns are magnified because the technique is so sensitive. Avoiding contamination is paramount for generating qPCR results that you can trust.
These are very basic tips that are common to both end-point and qPCR, but if you get these right, you are off to a good start no matter what your experimental goals are.
If you are looking for more information regarding qPCR, watch this supplementary video below.
We’re committed to supporting scientists who are using molecular biology to make a difference. Learn more about our qPCR Grant program.
Some thermostable DNA polymerases, including Taq, add a single nucleotide base extension to the 3′ end of amplified DNA fragments. These polymerases usually add an adenine, leaving an “A” overhang. There are several approaches to overcome the cloning difficulties presented by the presence of A overhangs on PCR products. One method involves treating the product with Klenow to create a blunt-ended fragment for subcloning. Another choice is to add restriction sites to the ends of your PCR fragments. You can do this by incorporating the desired restriction sites into the PCR primers. After amplification, the PCR product is digested and subcloned into the cloning vector. Take care when using this method, as not all restriction enzymes efficiently cleave at the ends of DNA fragments, and you may not be able to use every restriction enzyme you desire. There is some useful information about cutting with restriction sites close to the end of linear fragments in the Restriction Enzyme Resource Guide. Also, some restriction enzymes require extra bases outside the recognition site, adding further expense to the PCR primers as well as risk of priming to unrelated sequences in the genome.
Continue reading “A Quick Method for A Tailing PCR Products”
Have you ever thought about plant viruses? Unless you’re a farmer or avid gardener, probably not. And yet, for many people the battle against agricultural viruses never ends. Plant viruses cause billions of dollars in damage every year and leave millions of people food insecure (1–2), making viruses a major barrier to meeting the United Nations’ global sustainable development goal of Zero Hunger by 2030.
At the University of Western Australia, Senior Research Fellow Dr. Laura Boykin is using genomics and supercomputing to tackle the problem of viral plant diseases. In a recent study, Dr. Boykin and her colleagues used genome sequencing to inform disease management in cassava crops. For this work, they used the MinION, a miniature, portable sequencer made by Oxford Nanopore Technologies, to fully sequence the genomes of viruses infecting cassava plants.
Cassava (Manihot esculenta) is one of the 5 most important calorie sources worldwide (3). Over 800 million people rely on cassava for food and/or income (4). Cassava is susceptible to a group of viruses called begomoviruses, which are transmitted by whiteflies. Resistant cassava varieties are available. However, these resistant plants are usually only protected against a small number of begomoviruses, so proper deployment of these plants means farmers must know both whether their plants are infected and, if so, the strain of virus that’s causing the infection.
Continue reading “Moving Towards Zero Hunger, One Genome at a Time”In general, people like to know that their food is what the label says it is. It’s a real bummer to find out that beef lasagna you just ate was actually horsemeat. Plus, there are many religious, ethical and medical reasons to be cognizant of what you eat. Someone who’s gluten intolerant and Halal probably doesn’t want a bite of that BLT.
Labels don’t always accurately reflect what is in food. So how do we confirm that we are in fact buying crab, and not whitefish with a side of Vibrio contamination?
For the most part, it comes down to separation science. Scientists and technicians use various chromatographic methods, such as gas chromatography, liquid chromatography, and mass spectrometry, to separate the complex mixture of molecules in food into individual components. By first mapping out the molecular profile of reference samples, they can then take an unknown sample and compare its profile to what it should look like. If the two don’t match up, an analyst would assume that the unknown is not what it claims to be. Continue reading “Of Mice and Microbes: The Science Behind Food Analysis”
