Application Guide - QIAxcel Advanced

QIAxcel Advanced Application Guide 10/2016 79 Materials and Methods Amplification reactions (25 µl) were prepared with 20 mM Tris·HCl (pH 8.4), 50 mM KCl, 2.5 mM MgCl2 , 200 mM each dNTP, 2.5 µl primer, 40 ng genomic DNA, and 1.5 units Taq DNA polymerase (Life Technologies). Primers used to fingerprint repetitive ERIC sequences, ERIC 1R and ERIC 2, are described in (6). Samples were analyzed using the QIAxcel system together with the QIAxcel DNA High Resolution Kit and the 0M700 method with additional 120 second separation time. The QX Alignment Marker 15 bp/3 kb, the QX DNA Size Marker FX174/HaeIII, and bacteriophage lambda DNA digested with Eco47I (AvaII) (Fermentas) were included in the run. Results 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. PCR-amplified ERIC sequences of individual S. meliloti strains were resolved, exhibiting well separated, sharp bands in the range of 100–2000 bp. A representative gel image is shown in Figure 1. Results were compared with those from 6% poly (NAT) gel (Elchrom Scientific AG) analysis (data not shown). The binary call function of the BioCalculator Software was used to determine the presence or absence of specific fragments in the samples. DNA sizes were precisely and reproducibly calculated. The results were used in a later study to prepare a dendrogram displaying the relatedness of the isolated strains. 1 2 3 4 5 6 7 8 10 9 M Figure 1. Detection of genetic diversity for Sinorhizobium meliloti. PCR amplified ERIC sequences of individual S. meliloti strains were analyzed using the QIAxcel DNA High Resolution Kit. 1–9: Strains isolated from the nodules of alfalfa (Medicago sativa L.); 10: reference strain 2011; M: QX DNA Size Marker FX174/HaeIII. 1234567810

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