Viability PCR starter kits contain the materials that you need for selective detection of viable cells using either PMA or PMAxx™ viability dye and real-time PCR. All of the kits contain Forget-Me-Not™ qPCR Master Mix, and your choice of PMA or PMAxx™ dye.
Viability PCR originally used EMA (ethidium monoazide) to inactivate dead cell DNA. Biotium developed PMA (propidium monoazide) in collaboration with investigators at Montana State University (Nocker et al. 2006). PMA is more selective for dead cells than EMA, and became widely used for selective detection of viable microbes and viruses. PMAxx™ is Biotium’s newest viability PCR dye, designed to be more effective than PMA at eliminating PCR amplification of dead cell DNA. Therefore it provides the best discrimination between live and dead bacteria.
Both PMA and PMAxx™ are both offered as 20 mM solutions in water. We also offer PMA as a solid, and customers often ask whether it needs to be dissolved in DMSO. While in some publications users have prepared their PMA stock solution in DMSO, this is not necessary. Unlike the older viability dye EMA, which is not soluble in water, PMA and PMAxx™ are very water soluble.
Commercial halogen lamps (>600 W) for home use have been employed for photoactivating PMA in some publications, though results have not been consistent due to inevitable variation in the set-up configurations. If you decide to use a halogen lamp, we recommend that you lay tubes on a block of ice set 20 cm from the light source, on a rocking platform to ensure continuous mixing. Set the lamp so that the light source is pointing directly downward onto the samples (up to 45° downward slant is OK). Expose samples to light for 5-15 min.
Viability PCR starter kits contain the materials that you need for selective detection of viable cells using either PMA or PMAxx™ viability dye and real-time PCR. All of the kits contain Forget-Me-Not™ EvaGreen® qPCR Master Mix, and your choice of PMA or PMAxx™ dye. The user will need to supply their own primers to amplify their species of interest (see this blog post for primer design considerations).
This kit contains reagents sufficient to treat 80 bacterial cultures with PMA or PMAxx™, and perform 200 PCR reactions. There is an option to choose either PMA or PMAxx™ for the viability dye (see below for more information about these dyes). The number of samples that can be treated with PMA or PMAxx™ using the kit may vary depending on sample type. See the product protocol under the downloads tab and references for more information.
Store kit at -20 °C. After first thaw, Enhancer should be stored at 4°C. Protect PMA, PMAxx™ and Forget-Me-Not™ EvaGreen® qPCR Master Mix from light. Protect PMA and PMAxx™ from light during use. Components are stable for at least 6 months when stored as recommended. Components are stable for several freeze/thaw cycles.
PMA and PMAxx™ viability dyes
PMA is a high affinity photoreactive DNA binding dye developed by Biotium. The dye is weakly fluorescent by itself but becomes highly fluorescent upon binding to nucleic acids. It preferentially binds to dsDNA with high affinity. Upon photolysis, the photoreactive azido group on the dye is converted to a highly reactive nitrene radical, which readily reacts with any hydrocarbon moiety at the binding site to form a stable covalent nitrogen-carbon bond, thus resulting in permanent DNA modification. The dye is cell membrane-impermeable and thus can be used to selectively modify DNA from dead cells with compromised membrane integrity, while leaving DNA from viable cells intact. PMA inhibits PCR amplification of modified DNA templates by a combination of removal of modified DNA during purification and inhibition of template amplification by DNA polymerases. Consequently the dye is useful in the selective detection of viable pathogenic cells by quantitative real-time PCR.
PMAxx™ is a new and improved version of PMA designed by Biotium scientists to be a superior alternative to PMA. While PMA is generally effective at differentiating between live and dead bacteria by qPCR, it does not completely eliminate PCR products from dead cell DNA. This could potentially give false positive results. Biotium’s new dye PMAxx™ is much more effective at eliminating PCR amplification of dead cell DNA, and therefore provides the best discrimination between live and dead bacteria.
Forget-Me-Not™ EvaGreen® qPCR Master Mix
Forget-Me-Not™ qPCR Master Mix is a hot-start EvaGreen® dye-based master mix for use in real time PCR applications and DNA melt curve analysis. Forget-Me-Not™ master mix contains a low concentration of blue dye which allows you to see at a glance whether you forgot to add master mix to any of your tubes, so you can catch pipetting mistakes and avoid wasting time, reagents, and your precious DNA samples. It is formulated for qPCR using a fast cycling protocol, but also can be used for qPCR using regular cycling protocols. EvaGreen® dye is a unique DNA-binding dye with features ideal for both qPCR and melt curve analysis. EvaGreen® dye binds to dsDNA via a novel “release-on-demand” mechanism, which permits the use of a relatively high dye concentration in qPCR without PCR inhibition. Forget-Me-Not™ Master Mix contains Cheetah™ Taq, Biotium’s fast-activating chemically-modified hot-start Taq polymerase, which is particularly suitable for fast PCR cycling protocols.
Biotium sells two devices for the photolysis step of viability PCR. The PMA-Lite™ LED photolysis device is designed for PMAxx™- or PMA-treated samples in microcentrifuge tubes. The Glo-Plate™ Blue is a flat LED box ideal for samples in microplates, filters, or other formats.
Materials from Biotium are sold for research use only.
The Glo-Plate™ Blue LED Illuminator is a multi-functional blue LED light box. It can be used for photolysis of PMAxx™ or PMA in viability PCR. It is also an excellent way to develop the staining of gels stained with the visible blue DNA gel stain DNAzure®.