Genejet pcr purification kit

Two libraries of mutant MNA and MNB, respectively, were produced by error prone PCR using GeneMorph II Random mutagenesis kit (Agilent Technologies). By varying the template amount in the PCR reaction, the mutation frequency can be controlled. Mutation levels of 1–2 mutations/gene were desired and the primers used were epNGFPpQLinkNfor, epNGFPpQLinkNrev, epCGFPpQLinkNfor and epCGFPpQLinkNrev, with sequences as listed in Supplementary Information. To remove parent MNA from the NGFP-MNA-CGFP-MNB pQLinkN plasmid and to enable ligation of mutated MNA, the plasmid and the PCR product were separately digested at 37 °C with KpnI (Thermo Scientific) over night, purified with GeneJET PCR purification kit (Thermo Scientific) and digested with SacI (Thermo Scientific) at 37 °C for 1 h. For the last 15 min of the digestion, FastAP Thermosensitive alkaline phosphatase (Thermo Scientific) was added. To enable insertion of mutant MNB, the PCR product and the plasmid were separately digested at 37 °C with NdeI and PstI overnight and FastAP Thermosensitive alkaline phosphatase was added for the last 15 min. The digestion products were purified using GeneJET PCR purification kit (Thermo Scientific). The mutated MNA och MNB fragments were ligated into the appropriately digested plasmid by using T4 DNA ligase (NEB) for 16 h at 16 °C.

A solution of the fragmented DNA samples (250 ng) with an average length of 200 bp, 50 mM NaIO₄ in 50 mM sodium acetate buffer (pH 5.5) was incubated at 40 °C for 3 h followed by work-up with Micro Bio-Spin™ P-6 Gel Columns (Bio Rad) or a GeneJET PCR purification kit (Thermo Scientific). The resultant DNA solution (25 μL), 100 mM phosphate buffer (pH 6, 20 μL), 100 mM (+)-biotinamidohexanoic acid hydrazide (4 μL), and 100 mM p-anisidine (1 μL) were mixed and incubated at 40 °C for 4–12 h. The reaction mixture was purified by Micro Bio-Spin™ P-6 Gel Columns (Bio Rad) or a GeneJET PCR purification kit (Thermo Scientific). The samples were then subjected to NGS adapter ligation using reagents provided in the NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (New England Biolabs) and adapter solutions provided in the TruSeq DNA Sample Preparation Kit v3 (Illumina). Downstream procedures for base J enrichment were carried out as described in the chemical 5hmU pull-down sequencing procedure except for the use of KAPA HiFi HotStart Uracil + ReadyMix (KAPA Biosystems) as a PCR master mix.

MLST analysis was carried out as previously described [18 (link)]. In summary, fragments of seven housekeeping genes: arc, aroE, glpF, gmk, pta, tpi, and yqil (Table S1) were amplified following the protocol accessible at https://pubmlst.org/organisms/staphylococcus-aureus/primers (accessed on 1 April 2023). PCR products were purified with GeneJet PCR Purification Kit (Thermo Scientific) following the manufacturer’s specifications and sequenced by the Sanger Sequencing Service (Complutense University of Madrid, Spain). Clean sequences were queried in the database, and corresponding allele numbers were assigned. The combination of seven alleles gave the Sequence Type (ST) for each isolate. New alleles or STs were assigned when necessary by the international database of MLST for S. aureus. Phylogenetic analysis of the different STs was performed using the eBURST algorithm included in the software Phyloviz (http://www.phyloviz.net, accessed on 23 May 2023) and visualized in a minimum spanning tree.