Application Guide - QIAxcel Advanced

QIAxcel Advanced Application Guide 10/2016 83 with results from other band-based techniques (e.g., RFLP and PFGE sequencing data and binary and numerical character arrays). With these possibilities in mind, the combination of the QIAxcel Advanced System with BioNumerics was assessed as a method for pinpointing sources of pathogenic plant infections, such as bacterial canker of tomato. Materials and Methods The amplification reaction mixtures (25 µl) consisted of 1× PCR buffer (containing 100 mM Tris-HCl, 15 mM MgCl2 and 500 mM KCl at pH 8.3), 0.2 mM each of dNTPs, 0.5 µM primer (5’-TGCCGCCGCCGCC-3’), 0.5 U AmpliTaq DNA polymerase and 50–60 ng template DNA. DNA extracts were prepared according to Pitcher’s protocol (6) which was adapted for Gram-positive bacteria with an additional lysozyme step (5 mg lysozyme in 150 µl TE buffer per sample). The PCR program consisted of initial denaturation at 94°C for 5 min followed by 36 cycles (94°C for 5 min, 64°C for 45 s, 72°C for 2 min) and a final extension at 72°C for 10 min. Figure 1. Dendrogram and gel image showing the relatedness of Clavibacter michiganensis subsp. michiganensis strains. The primer in all cases was ISSR5 and the annealing temperature was 64°C. Year of isolation Strain – 1983 1 – 1962 2 – 1998 3 – 1996 4 – 1945 5 – 2006 6 – 1982 7 – 1984 8 – 1993 9 – 2007 10 – 2000 11 – 1990 12 – 1968 13 – 1967 14 – 1967 15 – ? 16 – 2012 17 – 2003 18 – 1978 19 – 1967 20 – 2008 21 – ? 22 – 19831 – 19622 – 19983 – 19964 – 19455 – 20066 – 19827 – 19848 – 19939 – 200710 – 200011 – 199012 – 196813 – 196714 – 196715 – 201217 – 200318 – 197819 – 196720 – 200821

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