Application Guide - QIAxcel Advanced

QIAxcel Advanced Application Guide 10/2016 21 PCR product (10 μl) was digested in a total volume of 40 μl with 10 U MboI (New England BioLabs) by incubating for 70 minutes at 37°C. The 518 bp wild-type fragment was digested into 3 fragments: 29 bp, 182 bp, and 307 bp. The pks3-7 mutant sequence is missing an MboI restriction site, and digestion of the mutant 518 bp fragment yielded 2 fragments: 211 bp and 307 bp. Digested samples were analyzed using the QIAxcel capillary electrophoresis system with the QIAxcel DNA Screening Kit and the AM320 method. The QX Alignment Marker 50 bp/500 bp and QX DNA Size Marker 50 bp–800 bp were included in the analysis. The QIAxcel capillary electrophoresis system processes samples in batches of 12 and allows analysis of up to 96 samples without manual intervention. The results can be displayed as an electropherogram as well as a gel-like image. Results and Discussion CAPS analyses were performed to screen the progeny of a backcross of pks3-7 against its isogenic wild-type control. By analyzing the F2 generation for the presence of the pks3-7 mutation using a specific CAPS marker, it was possible to follow the mutant allele through various crosses. The results of a CAPS analysis using the QIAxcel system are shown in Figure 1. In lanes 2, 4, and 6, DNA fragments from the wild type PKS3 gene are present (29 bp, 182 bp, and 307 bp). The fragments present in lane 3 (211 bp and 307 bp) indicate a homozygote for the pks3-7 mutation. The fragments in lanes 5, 7, and 8 (29 bp, 182 bp, 211 bp, and 307 bp) indicate heterozygosity for the pks3-7 mutation. The sizes of the DNA fragments estimated by BioCalculator Software are given in Table 1. Although the size differences between the fragments are small, the sharp banding patterns achieved using the QIAxcel system allowed more accurate size estimation than is possible with agarose gel electrophoresis (data not shown). Figure 1. CAPS analysis for the A. thaliana PKS3 gene. CAPS of the PKS3 gene were prepared from individual A. thaliana plants and resolved on the QIAxcel system using the QIAxcel DNA Screening Kit. 1: negative control; 2, 4, 6: wild type; 3: homozygous pks3-7 mutant; 5, 7, 8: heterozygote pks3-7 mutant; 9: QX DNA Size Marker 50 bp/800 bp. 1 2 3 4 5 6 7 8 9 123456789

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