Application Guide - QIAxcel Advanced

20 QIAxcel Advanced Application Guide 10/2016 A. thaliana genotyping with a CAPS marker for a pks3 mutant allele Martine Trevisan and Christian Fankhauser; Center for Integrative Genomics, University of Lausanne, Switzerland The QIAxcel® system was used for genotyping A. thaliana with CAPS markers, following mutations in the PKS3 gene (At1g18810), and to identify mutants in various crosses. Introduction Arabidopsis thaliana is a small flowering plant that is widely used as a model organism in plant cellular and molecular biology. Its genome sequence is known and is available through the Arabidopsis Information Resource (TAIR), as well as other sources, including seed stocks and collections of genetic and physical markers. The short life cycle (approximately 6 weeks from germination to seed maturation), enables highly efficient preparation of mutants for in-depth analysis of gene function. Once a mutant of interest has been identified, cleaved amplified polymorphic sequences (CAPS) are used to map the mutation (1). CAPS markers are also used to genotype known mutations. Previously, no mutations were known for the phytochrome kinase substrate protein 3 (PKS3) gene (At1g18810), a member of a small gene family in A. thaliana (2). The “targeting induced local lesions in genomes” (“tilling”) approach was used to identify a mutant in the PKS3 gene, pks3-7. Subsequently CAPS markers were used for genotyping, allowing the mutant to be followed in various genetic crosses. Materials and Methods Small arabidopsis leaves were homogenized by grinding in an Eppendorf® tube in 500 μl of 200 mM Tris (pH 7.5), 250 mM NaCl, 25 mM EDTA, and 0.5% SDS. After centrifugation, DNA was precipitated by adding equal amounts of isopropanol to the supernatant. After an additional centrifugation at 12,000 x g for 10 minutes, the DNA pellet was washed with 70% ethanol, air dried, and resuspended in 100–200 μl 10mM Tris, 1 mM EDTA (pH 8.0). PCR amplification was performed with Taq DNA polymerase (proprietary preparation) understandard reaction conditions in a 20 μl volume. PCR amplification using the CF523 (AAACA AGCCG ACATG GAACG) and CF524 (TCGTT ATGTT CTCAA TCTCG) primers yielded a prominent 518 bp fragment.

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