SYBR® Premix Ex Taq™ (Perfect Real Time) is a 2X concentration of premixed reagent which includes Takara’s Ex Taq™ HS and SYBR® Green I. The Ex Taq™ HS is an enzyme for hot start PCR which utilizes an anti-Taq antibody. The SYBR® Premix Ex Taq™ mix also contains dNTPs, Mg2+ and a newly developed buffer which provides superior specificity and increased amplification efficiency, thus, making it ideal for high-speed quantitative (also known as real-time) PCR (qPCR). In addition to 5 x 1.0 ml tubes of the 2X SYBR® Premix Ex Taq™, one tube (200 uL) of ROX reference dye (50X) is also supplied. (ROX is required by a few specific instruments, like ABI’s; Our Cepheid Smart Cycler does not require this reference dye.) One tube of SYBR® Premix Ex Taq™ contains 250 units of enzyme; 1 unit is the amount that will incorporate 10 nmol of dNTP into acid-insoluble products in 30 minutes at 74°C with activated salmon sperm DNA as the template-primer.
This premix is very easy to use. We just add 12.5 ul of the SYBR® premix to our template DNA (<100 ng), forward and reverse primers (0.2 uM). We then add PCR grade water to bring the volume to 25 uL and the reaction is ready to run. We have used this premix to develop standardized reaction conditions for primers which we are using for end-point PCR from listeria monocytogenes and E.coli templates. These primers worked very well with the SYBR® premix even when we multiplexed. In our multiplex experiment, we combined two different sets of primers and obtained two distinct melt curves and two different sizes of PCR product. We can easily discriminate our amplified product from our no template control by Tm as well as with the help of Ct.
The general amplification protocol which we use with this premix is as follows: 95ºC for 5-10 seconds. The fact that this kit does not require a prolonged denaturation is one of the features I like most; some SYBR® Green premixes require a 5 to 10 minute denaturation. The reduced denaturation time allows us to get the results in 45-53 minutes. After the initial denaturation, our samples cycle at 94ºC for 3 seconds, 60ºC for 20 seconds (annealing) and 72ºC for 30 seconds (extension). The results come as early before 25th cycle if the sample is positive. Our final interpretation includes analysis with the help of the melt curve.
I strongly prefer this product for the initial phase of standardization for newly synthesized primers which are based upon SYBR® Green chemistry.
Product Specialist
Molecular Diagnostics
Biotron