PCR is now a standard practice for all labs which are into molecular based examination. To get reproducible results, a lab should use well-standardized products which are sensitive and can provide accurate results. In our lab, we use and rely on Qiagen's HotStar Master Mix; this master mix gives us reproducible results with no non-specific amplicons.
HotStar Taq DNA Polymerase is a modified form of Qiagen's Taq DNA Polymerase. HotStar is supplied in an inactive state that has no polymerase activity at ambient temperature. This prevents extension of non-specifically annealed primers and primer-dimer pairs during PCR setup and even during initial PCR cycles. HotStar Taq is activated by a 15 minute incubation at 95°C. Qiagen provides the following components in the kit: HotStar Taq DNA Polymerase (5 units/ul), PCR buffer (provides final concentration of 1.5 mM MgCl2), MgCl2 solution, and Q solution. Q-Solution is a PCR additive that facilitates amplification of difficult templates by modifying the melting behavior of DNA. Although the kit has all of the other necessary reagents to perform amplification, it lacks dNTPs and DNase/RNase-free water.
I use this product in a specific application wherein the intended purpose is to detect 28 chromosomal alterations. Following is the brief protocol which we follow: First extract total RNA from whole blood and generate cDNA. Then comes the role of Qiagen Hotstart Taq polymerase. Reagent preparation is as follows: 5 ul of cDNA, 7 ul of the HotStar Taq and the appropriate amounts of 10X PCR buffer (44 ul), dNTP mix (9 ul), primers (11 ul), and ddH20 (205 ul). Final PCR reactions are 25 ul each. Cycling according to the following protocol is: initial activation at 95°C for 15 min, 25 cycles of 95°C for 30 sec/58°C for 30 sec/72°C for 90 sec then hold at 4°C
In summary, HotStar Tar DNA polymerase from Qiagen is an excellent product which in fact recommended by the kit manufacturer (Hemavision Full Kit from DNA Technology). They also mention this protocol is optimized for the use of HotStar Taq DNA Polymerase (Qiagen) only. Successful results using hot start DNA Polymerases from other vendors are not guaranteed.
Amplification products of 8 parallel multiplex RT-PCR reactions. The product with the higher molecular weight represents the internal positive control, and the lower bands represent the specific fusion genes.
hnHotStar Taq DNA Polymerase From Qiagen
Highly reproducible and specific results at an affordable price.
Additional dNTPs and PCR grade water must be supplied by the end-user.
The Bottom Line
I highly recommend this product for all gel PCR-based applications.