A 955 bp fragment was amplified from 100 ng of human genomic DNA using the HotStar HiFidelity Polymerase Kit and standard Taq DNA Polymerase. PCR products (4 µl each) were cloned using a commercially available TA or UA cloning kit. Blue-white screening was used to determine the cloning efficiency.
PCR was performed using HotStar HiFidelity DNA Polymerase (QIAGEN) and 4 high-fidelity PCR enzymes from the indicated suppliers. Parallel reactions were performed following the suppliers' recommendations, using 10 ng and 1 ng human genomic DNA. Two different high-fidelity enzymes from Supplier I were tested. A 2.3 kb fragment of the human IL9R gene was amplified in 40 PCR cycles. M: Markers.
Two different primer–template systems were amplified in duplicate using QIAGEN PCR Buffer and Taq DNA Polymerase in the absence (–) or presence (+) of 1x Q-Solution. Q-Solution enables specific amplification of difficult templates. [A] human angiotensin receptor II gene; [B] mouse protein kinase C gene; M: markers.
Ammonium and potassium cations in QIAGEN PCR Buffers increase specificity of primer annealing. K+ binds to phosphate groups (P–) on the DNA backbone, stabilizing the annealing of the primers to the template. NH4+, which exists both as the ammonium ion and as ammonia under thermal-cycling conditions, can interact with the hydrogen bonds between the bases (B), destabilizing the weak hydrogen bonds at mismatched bases. The combined effect of the two cations maintains a high ratio of specific-to-nonspecific primer-template binding over a wide temperature range.