Protocols for molecular cloning were adapted from Sambrook and Russell [11 ]. Escherichia coli strains DH5α (#18265017) and Stbl3™ (#C737303) were used for cloning (derived from chemically competent cells supplied by Thermo Fisher Scientific, Inc., Waltham, MA, USA). Preparation of electrocompetent cells and electroporation were performed at room temperature [12 (link)]. Electrocompetent E. coli and plasmid DNA were electroporated in 1 mm cuvettes at 1.8 kV using the MicroPulser electroporator (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Plasmid mini- and maxi-preparation protocols were adapted from Sambrook and Russell [11 ], with the addition of RNase A to cell lysis buffers. The QIAGEN-tip 500 was used to further purify crude plasmid extract (#10063; QIAGEN, Hilden, Germany).
Plasmid mini- and maxi-preparation protocols were adapted from Sambrook and Russell [11 ], with the addition of RNase A to cell lysis buffers. The QIAGEN-tip 500 was used to further purify crude plasmid extract (#10063; QIAGEN, Hilden, Germany).
