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 Taq Polymerase 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?”
Over the last few months we have published several blogs about qPCR—from basic pointers on avoiding contamination in these sensitive reactions to a collection of tips for successful qPCR. Today we look in depth at a paper that describes the design and and optimization of a qPCR assay, and in keeping with the season of winter in the Northern hemisphere, it is only fitting that the assay tests for the abundance and identity of ice-nucleating bacteria.
Ice-nucleating bacteria are gram-negative bacteria that occur in the environment and are able to “catalyze” the formation ice crystals at warmer temperatures because of the expression of specific, ice-nucleating proteins on their outer membrane. Ice-nucleating bacteria are found in abundance on crop plants, especially grains, and are estimated to cause one-billion dollars in crop damage from frost in the United States alone.
In addition to their abundance on crop plants, ice-nucleating bacteria are also found on natural vegetation and have been isolated from soil, snow, hail, cloud water, in the air above crops under dry conditions and during rain fall. They have even been isolated from soil, seedlings and snow in remote locations in Antarctica. For the bacteria, ice nucleation may be a method to promote dissemination through rain and snow.
Although ice-nucleating bacteria have been isolated from clouds, ice and rain, little is known about their true contribution to precipitation or other events such as glaciation. Are such bacteria the only source of warm-temperature (above temperatures at which ice crystals form without a catalyst) ice nucleation? Can they trigger precipitation directly? What are the factors that trigger their release from vegetation into the atmosphere? Can we determine their abundance and variety in the environment? Continue reading “Do you want to build a snowman? Developing and optimizing a qPCR assay to detect ice-nucleating activity”
In areas of the world where the electricity is intermittent, resources are limited and transporting bulky equipment and reagents that are sensitive to temperature fluctuations is difficult, diagnosis of viruses like dengue can be challenging. If you could reduce or eliminate the need for electricity dependent equipment for diagnostic assays without sacrificing sensitivity or specificity, it would be a boon to field workers. An article published in PLOS ONE describes how researchers developed a multiplex isothermal amplification method that could assess a potential dengue infection with a visual real-time or endpoint detection in a single tube. Continue reading “A Potential Single-Tube Multiplex Assay for Detecting Dengue Virus in the Field”
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.
10. Modify reaction buffer composition to adjust pH and salt concentration.
Continue reading “Top Ten Things You Can Do to Improve Your PCR Results”