4.5. Cloning and Sequencing of the PqHMGR Gene Promoter

The amplified DNA fragments were TOPO-TA cloned into a pCR^®IITOPO^® vector (Thermo Fisher Scientific, Waltham, MA, USA). Highly-efficient chemically-competent Escherichia coli DH5 alpha cells (New England Biolabs, Ipswich, MA, USA) were subjected to thermal shock and transformed by 5 μL of TOPO-TA cloning reaction product. The transformants were selected on Luria-Bertani medium (LB)-agar (2%) plates containing 30 μg·mL⁻¹ kanamycin sulphate (Genos, Lodz, Poland). The plasmid DNA was isolated from liquid E.coli cultures growing on LB medium in the presence of 30 μg·mL⁻¹ kanamycin sulphate [64]. The presence of an insert was verified by EcoRI digestion and subsequent 1% agarose gel electrophoresis. The selected clone was sequenced using a 3130xl Genetic Analyzer (Applied Biosystems, Waltham, MA, USA) at the CoreLab facility (Medical University of Lodz, Lodz, Poland). The sequencing process used M13 reverse and M13 forward primers possessing binding sites in the pCR^®IITOPO^® vector.