High pure pcr purification kit

PCR primers for the construction of IsaA protein-expressing plasmids are shown in Supplementary Table 1. DNA was isolated with the Genelute bacterial genomic DNA kit (Sigma-Aldrich, Zwijndrecht, The Netherlands). PCR was performed with the Phusion Hot Start II polymerase (Thermo Fisher Scientific, Wilmington, Delaware USA) using genomic DNA of S. aureus NCTC8325 as a template as described before^{34 (link)}. The PCR fragments purified using the High Pure PCR purification kit (Analytic Jena, Jena, Germany) were cleft with BamHI and NotI restriction enzymes (New England Biolabs, Ipswich, USA) and ligated to BamHI/NotI-linearized vector DNA using T4 DNA Ligase (New England Biolabs). Of note, PCR products obtained with primer combinations including a reverse primer with a stop codon (F1/R2) were inserted into plasmids pNG4110, and PCR products obtained with reverse primers lacking a stop codon (F1/R1) were inserted into plasmid pNG4210. The resulting plasmids were transferred to electrocompetent L. lactis PA1001 as described before³⁵ . All plasmids were verified by sequencing (Eurofins MWG Operon, Ebersberg, Germany).

Plasmids for the expression of the endolysins encoded by the phages bIL285 and bIL286 were constructed by generating PCR fragments of the genes pi252 and pi305, respectively (Phusion Hot Start II, Thermo Fisher Scientific, Wilmington, Delaware USA) by using L. lactis subsp. lactis IL1403 genomic DNA as a template (ZR Fungal/Bacterial DNA MiniPrep, Zymo Research, Irvine, CA, USA) with primers bil285F/bil286R and bil286F/bil286R. PCR fragment of pi252 was digested with BsaI to generate cloning overhangs and EcoRV to digest co-amplified pi149 and pi305 PCR fragments. PCR fragment pi305 was digested with BsaI, ApaI and AflII restriction enzymes (New England Biolabs, Ipswich, MA, USA) to generate cloning overhangs and digest co-amplified pi149 and pi252 PCR fragments, respectively. Both digested PCR fragments were inserted into NcoI/HindIII-linearized vector pNZ8048e. PCR products were purified by using the High Pure PCR Purification Kit (Analytic Jena, Jena, Germany). Ligations were performed by using T4 DNA Ligase (New England Biolabs), and the resulting plasmids were transferred to electrocompetent L. lactis PA1001 as described before (Leenhouts and Venema 1993 ). All selected plasmids were checked by sequencing (Eurofins MWG Operon, Ebersberg, Germany).