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”
One of the most critical parts of a Next Generation Sequencing (NGS) workflow is library preparation and nearly all NGS library preparation methods use some type of size-selective purification. This process involves removing unwanted fragment sizes that will interfere with downstream library preparation steps, sequencing or analysis.
Different applications may involve removing undesired enzymes and buffers or removal of nucleotides, primers and adapters for NGS library or PCR sample cleanup. In dual size selection methods, large and small DNA fragments are removed to ensure optimal library sizing prior to final sequencing. In all cases, accurate size selection is key to obtaining optimal downstream performance and NGS sequencing results.
Current methods and chemistries for the purposes listed above have been in use for several years; however, they are utilized at the cost of performance and ease-of-use. Many library preparation methods involve serial purifications which can result in a loss of DNA. Current methods can result in as much as 20-30% loss with each purification step. Ultimately this may necessitate greater starting material, which may not be possible with limited, precious samples, or the incorporation of more PCR cycles which can result in sequencing bias. Sample-to-sample reproducibility is a daily challenge that is also regularly cited as an area for improvement in size-selection.
We offer a wide array of GoTaq® DNA Polymerases, Buffers and Master Mixes, so we frequently answer questions about which product would best suit a researcher’s needs. On the product web page, you can filter the products by clicking the categories on the left hand side of the page to narrow down your search. Here are some guidelines to help you select the match that will best suit your PCR application. Continue reading “How Do I Choose the Right GoTaq® Product to Suit My Needs for EndPoint PCR?”
Are we better off now than we were 10 years ago? Often times this question is answered subjectively and will vary from person to person. We can empirically show how life expectancy has increased over the centuries thanks to advances in the fields of agriculture and medicine, but what about quality of life? Science affects our lives every day, and the general notion is that better science will (eventually) translate into better lives. There is a burning curiosity shared by myself and others to quantify how we have progressed in science over the years:
Bornmann and Mutz demonstrate in the image shown above how we have been doubling scientific output every nine years since the 1940s. That is not to say that we have become twice as smart or efficient; this phenomenon could be partially fueled by a desire to gain prestige through a high number of publications. To better assess the topic of efficiency, we can measure how long it takes to perform specific procedures and how much they cost. This article compares the rate of improvement for DNA sequencing, PCR, GC-MS and general automation to the rate of improvement for supercomputers and video game consoles.
Q: Can PCR products generated with GoTaq® DNA Polymerase be used to for T- vector cloning?
A: Yes. GoTaq® DNA Polymerase is a robust formulation of unmodified Taq Polymerase. GoTaq®DNA Polymerase lacks 3’ →5’ exonuclease activity (proof reading) and also displays non-template–dependent terminal transferase activity that adds a 3′ deoxyadenosine (dA) to product ends. As a result, PCR products amplified using GoTaq® DNA Polymerase will contain A-overhangs which makes it suitable for T-vector cloning.
Q: Can GoTaq® Long PCR Master Mix be used for T-Vector Cloning?
A: Yes it can. GoTaq® Long PCR Master Mix utilizes recombinant Taq DNA polymerase as well as a small amount of a recombinant proofreading DNA polymerase. This 3´→5´ exonuclease activity (proof reading) enables amplification of long targets. Despite the presence of a small amount of 3´→5´ exonuclease activity, the GoTaq® Long PCR Master Mix generates PCR products that can be successfully ligated into the pGEM®-T Easy Vector System.
We have demonstrated that GoTaq® Long PCR Master Mix successfully generated DNA fragments that could be ligated into pGEM®-T Easy Vector System without an A-tailing procedure, and with ligation efficiencies similar to those observed with the GoTaq® Green Master Mix.
For details refer to Truman, A., Hook, B. and Wieczorek, D. Using GoTaq® Long PCR Master Mix for T-Vector Cloning.
Tip: For cloning blunt-ended PCR fragments into T-vectors, use the A-tailing protocol discussed in the pGEM®-T and pGEM®-T Easy Technical Manual #TM042.
Q: How do I prepare PCR products for ligation? What products can be used to purify the DNA? Continue reading “T-Vector Cloning: Answers to Frequently Asked Questions”
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.
|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||Allows you to measure 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 allow quantitation of gene expression, measurement of DNA damage, and quantitation of viral load in a sample and other applications.
Anytime that you are preforming 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.
- Use aersol-resistant pipette tips, and have designated pipettors and tips for pre- and post-amplification steps.
- Wear gloves. Change them frequently.
- Have designated areas for pre- and post-amplification work.
- Use “master mixes” to minimize variability. A master mix is a ready-to-use mixture of your reaction components (excluding primers and sample) that you create for multiple reactions—because you are pipetting larger volumes to make it, and all of your reactions are getting their components from the same master mix, you are reducing variability from reaction to reaction.
- Dispense your primers into aliquots to minimize freeze-thaw cycles and the opportunity to introduce contaminants into a primer stock.
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.
The polymerase chain reaction (PCR) has revolutionized modern biology as a quick and easy way to generate amazing amounts of genomic data. However, when PCR doesn’t work, it can be frustrating. At these times, PCR and reverse transcription PCR (RT-PCR) inhibitors seem to be everywhere: They lie dormant in your starting material and can co-purify with the template of interest, and they can be introduced during sample handling or reaction setup. The effects of these inhibitors can range from partial inhibition and underestimation of the target nucleic acid amount to complete amplification failure. What is a scientist to do?
We decided to revisit a popular blog from our Promega Connections past for those of you in the amplification world. Enjoy:
- Modify reaction buffer composition to adjust pH and salt concentration.
- Titrate the amount of DNA polymerase.
- Add PCR enhancers such as BSA, betaine, DMSO, nonionic detergents, formamide or (NH4)2SO4.
- Switch to hot-start PCR.
- Optimize cycle number and cycling parameters, including denaturation and extension times.
- Choose PCR primer sequences wisely.
- Determine optimal DNA template quantity.
- Clean up your DNA template to remove PCR inhibitors.
- Determine the optimal annealing temperature of your PCR primer pair.
[Drum roll please]…and the most important thing you can do to improve your PCR results is:
- Titrate the magnesium concentration.
And if you want to, you can even build a custom PCR protocol using our iOS and Android device apps. Email it to your lab account, print it out for your notebook or just store it on your device for future reference.