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.
We have successfully cloned PCR products generated using GoTaq® and GoTaq® Flexi DNA Polymerases into the pGEM®-T (Cat.# A3600), pGEM®-T Easy (Cat.# A1360) and pTARGET™ (Cat.# A1410) Vectors.
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.
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?
Yesterday I listened in on the Webinar “Getting the Most Out of Your DNA Analysis from Purification to Downstream Assays”, presented by Eric Vincent–a Product Manager in the Promega Genomics group.
This is the webinar for you if you have ever wondered about the relative advantages and disadvantages of the many methods available for DNA purification, quantitation and analysis, or if you are comparing options for low- to high-throughput DNA purification. Eric presents a clear analyses of each of the steps in a basic DNA workflow: Purification, Quantitation, Quality Determination, and Downstream Analysis, providing key considerations and detailing the potential limitations of the methods commonly used at each step.
The DNA purification method chosen has an affect on the quality and integrity of the DNA isolated, and can therefore affect performance in downstream assays. Accuracy of quantitation also affects success, and the various downstream assays themselves (such as end-point PCR, qPCR, and sequencing) each have different sensitivities to factors such as DNA yield, quality, and integrity, and the presence of inhibitors. Continue reading “DNA Purification, Quantitation and Analysis Explained”
Over the years we have produced several articles and technical resources on PCR. One of the most widely used is a chapter on PCR in the Protocols and Applications guide, featuring an explanation of the techniques involved, tips for reaction optimization, and example protocols for routine PCR, qPCR and RT-PCR. The protocols and applications guide is part of the Promega App, available on the promega.com web site, and as an iPhone/iPad and Android App.
Recently, we released an update to the Android app that includes customizable PCR protocols. Right now, three protocols are available in this format (Basic PCR, Hot-Start PCR and qPCR). These custom protocols allow you to input specific values for your experiment, such as concentration of stock solutions, desired number of reactions, etc. These values are used to calculate the volumes required at each step in the procedure, and a customized protocol is generated with the volumes of each reagent calculated and incorporated. Once generated, protocols can be saved for re-use, or emailed to an account for subsequent printing or incorporation into a lab notebook. Continue reading “PCR Protocols for Android”
There are several possible reasons why the PCR product may not be recovered after ligation, bacterial transformation and plating when using the pGEM®-T or pGEM®-T Easy Vector Systems.
The PCR fragment may not be A-tailed. Without the A overhangs, the PCR product cannot be ligated into a T vector. Use a nonproofreading DNA polymerase like GoTaq® DNA Polymerase for PCR. If a proofreading DNA polymerase is used, A overhangs will need to be added. Purify the PCR fragment, and set up an A-tailing reaction (see the pGEM®-T and pGEM®-T Easy Vector Systems Technical Manual #TM042). The A-tailed product can be added directly to the ligation as described in the pGEM®-T or pGEM®-T Easy Vector protocol.
The insert:vector ratio may not be optimal. The ideal ratio for each insert to a vector can vary. For example, the Control Insert DNA works well at a 1:1 ratio, but another insert may be ligated more efficiently at a 3:1 ratio. Check the integrity and quantity of your PCR fragment by gel analysis. Optimize the insert:vector ratio (see Technical Manual #TM042).
Multiple PCR products were amplified and cloned into the pGEM®-T or pGEM®-T Easy Vector. Other amplification products including primer dimers will compete for ligation into the T vector, decreasing the possibility that the desired insert will be cloned. To minimize other competing products, gel purify the PCR fragment of interest.
Promega Technical Services Scientists are here to assist you in troubleshooting your experiments at any time. Contact Technical Services.
Since the first photograph of bats with white muzzles in Albany, NY, was published, hibernating bat populations in the northeastern U.S. have been devastated by an emerging disease, White-Nose Syndrome (WNS), which continues to spread throughout the United States and now has been found in two Canadian provinces. Bats suffering from WNS are emaciated with little or no body fat and have a characteristic white fungal growth on their wing membranes, ears and muzzles. Instead of hibernating all winter, these bats can be seen active in the snow, when there is virtually no food available.
The infectious agent that responsible for WNS is a new species of the cold-loving fungus, Pseudogymnoascus destructans (formerly, Geomyces destructans) . Currently, WNS is confirmed either through histological analysis or by fungal culture. Both of these techniques have significant limitations. First, they have turnaround times of at least one week. Secondly, they require large amounts of tissue sample from affected bats, more than can be reasonably taken from live bats. Histological analysis is a laborious process that requires highly skilled and trained personnel. Fungal culture can be difficult because bats harbor many bacteria and fungi, and getting a pure culture of a causative organism is not simple. Furthermore, researchers need a quick method for assessing spread of the disease that can provide results quickly.
Polymerase Chain Reaction (PCR), already used for a host of diagnostic tests in humans, plants and animals, is a logical choice. In a paper published in the Journal of Veterinary Diagnostic Investigation, Lorch and colleagues design and evaluate a PCR-based diagnostic method for WNS. They compare the PCR method to a fungal culture method and the “gold standard” traditional histological analysis. Continue reading “PCR-Based Diagnosis Wins by a Mile In the White-Nose Syndrome Race”
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