Multiplex PCR of 16 targets (99–955 bp) was carried out for 35 cycles using standard conditions for the QIAGEN Multiplex PCR Kit, without further optimization or using a variety of conditions with a hot-start DNA polymerase from Supplier AII. [A] Comparison using 2.5 units per 50 µl reaction of the hot-start DNA polymerase from Supplier AII and with the indicated Mg2+ concentrations. [B] Comparison using the optimized Mg2+ concentration (3.5 mM) for the hot-start DNA polymerase from Supplier AII (part A) and the indicated amounts of enzyme per 50 µl reaction. Successful results were ensured with the QIAGEN Multiplex PCR Kit. M: markers.
Transgenic mice were screened using the QIAGEN Multiplex PCR Kit and sets of 3 primers to distinguish wild-type (wt), heterozygous mutant (ht), and homozygous mutant (hm) mice. [A] Using a primer set for the recombination activating gene 2 locus. [B] Using a primer set for the interferon-γ gene locus. M: markers. (Data kindly provided by S. zur Lage and S. Weiss, National Research Center for Biotechnology, Braunschweig, Germany.)
Analysis of microsatellite loci D3S1358, TH01, D21S11, D18S51, and Penta E was carried out using 1 ng of K562 human genomic DNA and fluorescein-labeled primers. Reactions were analyzed on the ABI PRISM 377 Sequencer. Top: The QIAGEN Multiplex PCR Kit ensured high sensitivity and uniform signal intensity. Bottom: Results using a hot-start DNA polymerase from Supplier AII.
The Multiplex PCR Buffer ensures stable and efficeint primer annealing. [A] NH4+ ions prevent nonspecific primers from annealing to the template. [B] Synthetic factor MP, an innovative PCR additive, increases the local concentration of primers at the template. Together with K+ and other cations, synthetic factor MP stabilizes specifically bound primers, allowing efficient primer extension by HotStarTaq DNA Polymerase.