PRW LINE-like mapping was done in Jaén, Spain. 1 µg of PRW LINE-like DNA was labelled by nick translation using the biotin-NT-mix (Roche, Basel, Switzerland). The probe was then precipitated along with 50 μg yeast RNA and 50 μg salmon sperm DNA and redissolved in 50% formamide in 2 × SSC. Fluorescence in situ hybridization (FISH) was performed according to the published protocol39 (link) with listed modifications40 (link).
PRW Bel-Pao mapping was done in České Budějovice, Czech Republic. Both fragments (I and II, which do not contain the deleted region—see below) were labelled by PCR containing 0.04 mM each of dATP, dCTP and dGTP; 0.014 mM dTTP, 0.025 mM biotin-16-dUTP (Jena Bioscience, Jena, Germany), 1× ExTaq buffer (TaKaRa), 1–10 ng plasmid DNA, 1 μM of each primer and 2U DNA ExTaq Polymerase (TaKaRa) under the same conditions as described above. FISH was performed according to the published protocol41 (link), with signal amplification using Cy3-conjugated streptavidin (Jackson ImmunoRes. Labs. Inc, West Grove, PA, USA) at a dilution of 1:1000 with washing blocking buffer.
PRW Bel-Pao mapping was done in České Budějovice, Czech Republic. Both fragments (I and II, which do not contain the deleted region—see below) were labelled by PCR containing 0.04 mM each of dATP, dCTP and dGTP; 0.014 mM dTTP, 0.025 mM biotin-16-dUTP (Jena Bioscience, Jena, Germany), 1× ExTaq buffer (TaKaRa), 1–10 ng plasmid DNA, 1 μM of each primer and 2U DNA ExTaq Polymerase (TaKaRa) under the same conditions as described above. FISH was performed according to the published protocol41 (link), with signal amplification using Cy3-conjugated streptavidin (Jackson ImmunoRes. Labs. Inc, West Grove, PA, USA) at a dilution of 1:1000 with washing blocking buffer.
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