Abi prism 3100 avant genetic analyzer

DNA was extracted from peripheral blood samples by using the Chemagic MSM I automated system (Chemagen, Baesweiler, Germany). Microsatellite markers D2S148, D2S2173, D2S324 and D2S2310 were amplified using fluorescently-labeled primers and PCR conditions as previously reported [30 (link)]. Amplified alleles were resolved by capillary electrophoresis in an ABI Prism 3100 Avant Genetic Analyzer (Applied Biosystems, Waltham, MA, USA). Primers and conditions for PCR amplification of all seven exons of the PJVK gene are shown in Table 1. Sanger DNA sequencing was performed in an ABI Prism 3100 Avant Genetic Analyzer (Applied Biosystems, Waltham, MA, USA).

Ten microsatellite markers were used for the analysis [38 (link),39 (link),40 (link)]. The PCR mix contained 50 ng of genomic DNA, 0.25 µM of each primer, 0.2 mM each dNTP, 2 mM MgCl₂, 1 × Euroclone reaction buffer and 2 U EuroTaq DNA polymerase (Euroclone^®, Milan, Italy) in a total volume of 25 µL. The forward primer was labeled with FAM and HEX fluorochromes (Sigma-Aldrich, St. Louis, MO, USA). PCR reactions were performed in a C1000™ Thermal Cycler (Bio-rad, Hercules, CA, USA) under the following conditions: 94 °C for 5 min, 35 cycles at 94 °C for 30 s, 50 to 60 °C (depending on the SSR primer combination) for 30 s, and 72 °C for 30 s, and final elongation at 72 °C for 60 min. PCR products were separated using the ABI PRISM 3100 Avant Genetic Analyzer (Life Technologies, Carlsbad, CA, USA), using a mixture of 2 µL PCR reaction, 12 µL Hi-Di™ formamide (Life Technologies, Carlsbad, CA, USA), and 0.3 µL GeneScan™ 500 ROX™ size standard (Life Technologies, Carlsbad, CA, USA). Allele size was determined using GeneMapper^® software version 3.7 (Applied Biosystems, Foster City, CA, USA).

All dogs included in this study were previously referred for MCTs to the Animal
Medical Center at Tokyo University of Agriculture and Technology. After
appropriate surgical removal or fine needle aspiration of MCT specimens, total
RNA from each sample was extracted by using Isogen (Nippon Gene, Toyama, Japan)
and cDNA was synthesized with PrimeScript (Takara, Otsu, Japan). Polymerase
chain reaction (PCR) was performed using a c-kit-specific forward primer
(5′-GGA ATT CGC CAC CGC GAT GAG AGG CGC TCG CGG CGC
CT-3′), a c-kit-specific reverse primer (5′-CTC TGC
GGC CGC TCA CAC ATC TTC GTG TAC CAG CA-3′), and PrimeSTAR Max DNA
Polymerase (Takara), according to the manufacturer’s instructions.
The c-kit gene was sequenced using the BigDye Terminator v3.1 Cycle
Sequencing Kit (Life Technologies, Gaithersburg, MD), forward primers
corresponding to bases 243–263, 648–667,
1050–1069, 1484–1504, 1843–1862,
2205–2225, and 2639–2657, and a reverse primer
corresponding to bases 386–406 (GenBank accession no. AF044249).
Samples were analyzed using the ABI PRISM 3100-Avant Genetic Analyzer
(Life Technologies). All experiments using clinical samples complied with the
standards specified in the guidelines of the University Animal Care and Use
Committee of the Tokyo University of Agriculture and Technology.