The TaqMan® Universal PCR Master Mix combines components of the TaqMan® PCR Core Reagents Kit with additional key components in one easy-to-use premix. Applied Biosystems has optimized these proprietary buffer components and stabilizers to enhance reaction performance across all sample types, even with the most demanding G/C-rich templates. It contains a passive internal reference, which minimizes well-to-well variability that can result from a variety of causes, such as pipetting errors and sample evaporation. The passive reference is also essential for accurate results when multiple probes (with different reporter dyes) are combined in a single tube. Enzyme concentration is also optimized to give superior performance in multiplex assay systems. Input sample template is DNA or cDNA. The fluorescent signal can be collected with ABI PRISM® 7700 or 7900HT Sequence Detection System instruments.
The Taqman® Master Mix also contains an enzyme, AmpErase® Uracil N-glycosylase (UNG), which protects against carry-over contamination. In this PCR mix, dTTPs are substituted for dUTPs. Consequently, products from PCR amplification contain uracil, and are readily distinguishable from native thymidine-containing DNA templates. UNG eliminate products from previous PCR amplifications by excising uracil residues and degrading the resulting abasic polynucleotide with heat. Therefore, only native nucleic acid templates are being PCR amplified.
The TaqMan® Universal PCR Master Mix is optimized for 5’ nuclease assay using TaqMan® probes and contains AmpliTaq Gold® DNA Polymerase, dNTPs with dUTP, Passive Reference 1, optimized buffer components, and AmpErase® UNG. It is available in several sizes and is supplied at 2X concentration. Recommended storage conditions are 2-8°C, so that thawing is not required before use.
I use the TaqMan® Universal PCR Master Mix for real-time PCR using custom TaqMan® probes (Applied Biosystems). The reaction set-up includes combining, in an optically clear plate, PCR master mix, cDNA template, unlabeled PCR primers and fluorescent TagMan® MGB probe. For thermal cycling I use optimal annealing temperatures calculated for my primers by the primer design computer program. It is important to remember that an extra step at 50 o C needs to be added to the cycling program for UNG activation. Remarkably, nearly all of my experiments worked well the first time with great reproducibility.
However, it is important to note that TaqMan® Universal PCR Master Mix reagent is expensive. If financing is limited, it would be prudent to look for alternative suppliers. Just as Tagman® probes that are very expensive are now available from other suppliers much cheaper, I assume that each of the reagents (or even all of them combined) could be obtained from other suppliers for less.
In summary, TaqMan® Universal PCR Master Mix, No AmpErase® UNG is an easy-to-use solution contains all components needed for all 5' nuclease assays using TaqMan® probes to minimize assay set-up time, but it is expensive.
Yelena Bykhovskaya, PhD
Research Scientist I
Molecular Hematology Laboratory
Cedars-Sinai Medical Center