Phusion high delity dna polymerase

In order to reliably identify healthy, carrier and affected animals, a PCR-based genetic test was developed. The F4R4 pair of primers corresponding to the 5' end of intron 20 was used to amplify the WT allele. A pair of primers were designed to amplify across the distant breakpoint, named mutF-mutR (normal size 1939 bp) and supposed to amplify the WT allele but not the LRMD. PCR was set up using the Phusion high delity DNA polymerase (Thermo Scienti c ®). The LRMD F4-mutR and mutF-R4 bands were extracted from the gel using a Nucleospin Gel and PCR Cleanup kit (Macherey-Nagel), and Sanger sequencing was performed in order to characterise the mutation breakpoints. The diagnostic PCR was designed to be run with three primers in a single PCR: F4, R4 (1661 bp band WT allele), and mutR (890 bp band LRMD allele).

All of the restriction enzymes, Phusion high-delity DNA polymerase and T4 DNA ligase were purchased from Thermo Scienti c (USA). The reactions followed standard protocols as described by manufacturers. The Plasmid Mini Kit, Gel Out extraction kit and Genomic Mini AX Yeast Spin kit were obtained from A&A Biotechnology (Poland). Isolation of plasmid DNA, DNA from gel puri cation and gDNA extraction followed protocols supplied by the manufacturer. E. coli transformation followed the standard chemical protocol with selective medium containing ampicillin to plate Y. lipolytica strains, which were transformed with overexpression cassettes or a deletion cassette by the lithium acetate method. Transformations resulted in strains AJD ΔB07117g and AJD pAD-B07117g. Additionally the strain AJD ΔB07117 was transformed with an overexpression cassette resulting in the strain AJD-c-B07117g.