Application Guide - QIAxcel Advanced

QIAxcel Advanced Application Guide 10/2016 35 Standard amplification reactions (25 µl) were prepared using 0.4 µM of each primer. After initial denaturation at 95°C for 5 minutes, reactions were subjected to 35 cycles of 95°C, 62°C, and 72°C for 30 seconds each. Reactions were incubated for a final elongation at 72°C for 5 minutes. Samples were analyzed using the QIAxcel system with the QIAxcel DNA Screening Kit and the AL320 method. The QX Alignment Marker 15 bp/1 kb and the QX DNA Size Marker 50–800 bp were included in the analysis. Results and Discussion The QIAxcel system was used to detect the presence of the Cre transgene in putative transgenic mice. Figure 1 shows the results of genotyping in which the Cre-specific DNA fragment (320 bp, present in lanes 2, 6, 11, and 12) as well as the control fragment from the myogenin gene (250 bp, present in all lanes) were unambiguously identified. The QIAxcel capillary electrophoresis system processes samples in batches of 12 and allows analysis of up to 96 samples without manual intervention. Results can be displayed as a gel-like image as well as an electropherogram. Using the BioCalculator Software, the presence of specific DNA fragments as well as their sizes were accurately determined. Cre transgenic mice will be used in breeding for knockout functional analysis. Figure 1. Detection of the Cre transgene in mice. DNA was isolated from mouse ear tissue and subjected to PCR amplification using primers for the Cre transgene as well as for a myogenin control. PCR products were analyzed on the QIAxcel system using the QIAxcel DNA Screening Kit. The 250 bp control fragment amplified from the myogenin gene is present in all lanes, indicating that amplification reactions were successful. The 320 bp fragment present in lanes 2, 6, 11, and 12 indicates the presence of the Cre transgene in these mice. 1 3 5 7 9 11 2 4 6 8 10 12 1357911 24681012

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