FAQ-1047
What is the largest PCR amplicon that can be amplified with the HotStar HiFidelity Polymerase Kit?
In QIAGEN labs, we have amplified PCR products up to 5 kb from complex genomic DNA, and up to 10 kb from less complex lambda phage DNA with the HotStar HiFidelity Polymerase Kit, following standard protocols in the HotStar HiFidelity PCR Handbook.
For targets larger than 5 kb of complex genomic DNA, and larger than 10 kb of less complex DNA, we recommend to follow the protocol 'Amplification of Long PCR Products' in the HotStar HiFidelity PCR Handbook. The protocol uses a mixture of HotStar HiFidelity DNA Polymerase and Taq, or HotStar Taq Plus DNA Polymerase, and allows much longer fragments to be generated. In-house we have tested fragments up to 13 kb from complex genomic DNA or up to 30 kb from less complex lambda phage DNA using this protocol.
Related products
HotStarTaq Plus DNA Polymerase
The polymerase combines the high specificity, sensitivity, and minimal optimization of HotStarTaq DNA Polymerase, with a fast 5-minute activation time. PCR can be set up at room temperature and reactions can be directly loaded onto a gel, due to novel CoralLoad PCR Buffer, which contains two gel-tracking dyes. The standard QIAGEN PCR Buffer is also included for greater convenience. In addition, Q-Solution, a novel additive that enables efficient amplification of "difficult" (e.g., GC rich) templates, is also provided. The unique kit components and optimized protocols streamline the PCR procedure.
Taq DNA Polymerase
Taq DNA Polymerase is a replicative polymerase derived from the thermophilic eubacterium Thermus aquaticus. Thermostable activity at temperatures above 70°C makes the enzyme suitable for standard and specialized PCR amplification applications. QIAGEN Taq DNA Polymerase is supplied with PCR Buffer specially formulated for fast setup with minimal optimization of PCR parameters, Q-Solution to facilitate amplification of "difficult" (e.g., GC-rich) templates and the additional time-saving advantage of CoralLoad PCR Buffer with two gel-tracking dyes to enable immediate loading of PCR products.
QIAGEN Taq DNA Polymerase is also available in Taq PCR Core Kit that includes dNTPs. If you prefer a Master Mix format, including all needed components, go to Taq PCR Master Mix Kit.
The source of Taq DNA polymerase, the thermophilic eubacterium Thermus aquaticus, was first identified in 1966, living and thriving at about 75°C in the waters of a hot spring in Yellowstone National Park. Taq DNA polymerase has a temperature optimum of 72°C and does not denature at 95°C. This intrinsic thermostability makes Taq DNA polymerase suitable for standard and specialized PCR amplification applications based on its core competence to maintain relatively high catalytic activity and stability after multiple rounds of thermal cycling at high temperatures.
Taq DNA polymerase has 5’→3’ exonuclease activity to remove RNA primers but lacks 3’→5’ exonuclease “proofreading” activity. Many DNA polymerases perform highly accurate DNA synthesis even in the absence of exonucleolytic proofreading. In nature, Thermus aquaticus compensates for the lack of proofreading with a mismatch repair (MMR) system including a MutS homolog that plays a crucial role in correcting replication errors. Taq DNA polymerase performs best when amplifying DNA fragments <2 kb but can amplify longer fragments efficiently under defined reaction conditions — dNTP concentration, pH and the concentration of MgCl2 relative to the total concentration of dNTPs present. Under these conditions, an error rate for Taq DNA polymerase per nucleotide polymerized at 70°C can be achieved as low as 10-5 for base substitution errors and 10-6 for frameshift errors.
The non-template-dependent terminal transferase activity inherent in Taq DNA polymerase and other nonproofreading DNA polymerases provides a highly efficient method to clone PCR products. Taq DNA polymerase adds a single, unpaired residue, preferentially an adenosyl residue, to each 3'-end of a double-stranded amplified product (extra A addition). This property is an advantage for the TA-cloning strategy but may present drawbacks when Taq DNA polymerase is used for microsatellite genotyping analysis.
QIAGEN Taq DNA Polymerase is designed to deliver fast, reliable PCR-related workflows that are easily optimized for efficient amplification performance and reduced error rates.
If you are looking for a DNA polymerase with higher fidelity or longer range than Taq DNA Polymerase, explore our long range or higher fidelity enzymes and mixes. Long range PCR provides highly sensitive and specific long-range amplification for up to 30 kb using any DNA or cDNA template. High fidelity enzymes and master mixes confer higher fidelity, speed and performance compared to standard Taq DNA Polymerase.