Higene gel pcr purification system

The human DNMT1 gene was amplified through polymerase chain reaction (PCR) using Pfu polymerase (Biofact, PD302-50h). Primers were screened with Primer3web version 4.1.0 and synthesized by Bioneer Corporation (Korea). The amplicon and primer sequences for DNMT1 are noted in Fig. S1. The PCR products were purified with a HiGene Gel & PCR Purification system (Biofact, GP104-100), and cloned into an All in One PCR Cloning vector (Biofact, VT201-020). The cloned vector constructs were extracted using an NucleoBond Xtra Midi kit (Machery-Nagel, 740410.50).

Standard cloning procedures were followed in this study (Sambrook et al., 1989 ). Oligonucleotides were purchased from Macrogen Co., Ltd., Korea and all the Oligonucleotides used in this study are given in Table 2. The genomic DNA from A. nosocomialis and the plasmid DNA from E. coli were isolated using the PureHelix™ Genomic DNA Prep Kit (Nanohelix Co. Ltd., Korea) and AccuPrep® Plasmid Extraction Kit (Bioneer Corp., Korea), respectively. The plasmids were introduced into E. coli and A. nosocomialis by heat shock method (Hanahan, 1983 (link)) and conjugation (Oh et al., 2015 (link)), respectively. PrimeSTAR GXL Taq DNA polymerase (TaKaRa, Japan) was used for polymerase chain reaction (PCR) amplification of DNA fragments. The DNA fragments were purified or eluted from gel using HiGene™ Gel & PCR Purification System (Biofact Co., Ltd., Korea). Restriction and DNA modifying enzymes were purchased from New England Biolabs, USA. The nucleotide sequence of all recombinant strains and mutants were confirmed by sequencing (Macrogen Co., Ltd., Korea).