Genomic DNA was isolated from freeze-dried mycelium using the method described previously [27 (link)]. PCR amplification of SSRs was carried out with the proofreading Phusion® High-Fidelity DNA polymerase (Thermo Scientific). The primers used in this study are listed in S2 Table . PCR products were cloned into the Escherichia coli plasmid vector pGEM®-T Easy (Promega) as per manufacturer’s instructions. Plasmids were transformed into E.coli DH5α chemically competent cells, the plasmids purified and the sequences of the SSRs determined. DNA sequencing was performed by the Massey Genome Service using BigDyeTM Terminator (version 3.1) Ready Reaction Cycle Sequencing Kit (Applied Biosystems). Sequence analysis was performed using MacVector® version 10.0.2 (MacVector Inc.).
Bigdye terminator version 3.1 ready reaction cycle sequencing kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The BigDye Terminator (version 3.1) Ready Reaction Cycle Sequencing Kit is a reagent system for DNA sequencing. It contains the necessary components for performing DNA sequencing reactions.
Automatically generated - may contain errors
Lab products found in correlation
Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (2 mentions)
Abi 3730 genetic analyzer, by Thermo Fisher Scientific (2 mentions)
Pgem t easy, by Promega (1 mentions)
Abi 3730 genetic analyser, by Thermo Fisher Scientific (1 mentions)
High pure plasmid isolation kit, by Roche (1 mentions)
Taq dna polymerase, by Roche (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
4 protocols using bigdye terminator version 3.1 ready reaction cycle sequencing kit
1
Isolation and Sequencing of Fungal Microsatellites
2
Molecular Techniques for Microbial DNA Extraction and Analysis
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S. cerevisiae plasmid DNA was extracted as previously described (Colot et al.,2006 ). E. coli plasmid DNA was extracted using the High Pure Plasmid Isolation Kit (Roche, Indianapolis, IN, USA). Fungal DNA was extracted as previously described (Byrd et al., 1990 ). Cloning and deletion mutant screening PCR reactions were performed using Phusion® High Fidelity DNA Polymerase (Thermo Scientific, Waltham, MA, USA) and Taq DNA polymerase (Roche), respectively. Sequencing reactions were performed using the Big‐DyeTM Terminator Version 3.1 Ready Reaction Cycle Sequencing Kit (Applied BioSystems, Carlsbad, California, USA) and separated using an ABI3730 genetic analyser (Applied Bio Systems). Sequence data were assembled and analysed using MacVector sequence assembly software, version 12.0.5.
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S. cerevisiae plasmid DNA was extracted as previously described (Colot et al.,
3
Sanger Validation of Pathogenic Variants
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The identified pathogenic and likely pathogenic variants were validated using Sanger sequencing, which was carried out using BigDyeTM Terminator Version 3.1 Ready Reaction Cycle Sequencing Kit on an ABI3730 Genetic Analyzer (Applied Biosystems, Waltham, CA, USA). The sequencing template PCR conditions and sequencing primers are presented in Table S2 .
4
Detecting FASL Variant in NZ Cats
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To detect the presence of the variant in FASL in the archived samples from NZ cats, specific primers were designed to amplify a 173 bp region of DNA that contained the variant using Geneious 8.1 software (Biomatters Ltd, Auckland, NZ; Table 1). Amplification conditions were 95 °C for 10 min followed by 40 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 60 s. The final extension was at 72 °C for 5 min. Amplified DNA was purified from a 1.5 % agarose gel containing ethidium bromide using a Qiaquick PCR purification kit (Qiagen) and submitted for automatic dye-terminator cycle sequencing with BigDye TM Terminator Version 3.1 Ready Reaction Cycle Sequencing kit and using a ABI3730 Genetic Analyzer (Applied Biosystems Inc, California, USA). The presence of the variant within DNA extracted from blood samples taken from Australian cats was investigated using different specific primers that amplified a 659 bp fragment (Table 1). PCR amplification, purification, and sequencing were performed as described previously (Gandolfi et al. 2015) .
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