SphI NEB#R0182

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	The amplified product of 246 bp was digested with the restriction enzymes Xhol and Sphl, and purified by gel extraction kit.

Protocol tips
The amplified product of 246 bp was digested with the restriction enzymes Xhol and Sphl, and purified by gel extraction kit.

Publication protocol

The merF gene (wild type) of highly mercury resistant Enterobacter sp. AZ-15 was amplified using the primers given in Table ​Table1.1. The amplified product of 246 bp was digested with the restriction enzymes Xhol and Sphl, and purified by gel extraction kit. The digested merF gene was ligated with the Xhol-Sphl-cleaved expression vector pET31b(+) (Kuliopulos et al., 1987). The genetic codons encoding cysteine residues in the native sequence of merF gene were mutated into codons encoding serine residues using site directed mutagenesis kit by designing two sets of primers (Table ​(Table1).1). Both sequences of merF gene (merF wild type and mutated merF) along with translated peptide sequences were submitted to the NCBI database (www.ncbi.nlm.nih.gov/) and obtained accession numbers as MF185183 and MF185184, respectively. The designed recombinant plasmid containing mutated merF (merFm) gene was transformed into DH5α competent cells and identified by using same restriction enzymes on agarose gel. The nucleotide sequence of recombinant pET31b(+) containing merFm gene was confirmed by DNA sequencing facility provided marketplace, University of California, San Diego (USA). Then the recombinant plasmid pET31b(+) was transformed into C43(DE3) competent E. coli cells. After transformation, stable C43(DE3): pET31b+merFm clones were screened and stored as glycerol stocks (20% of glycerol) at −80°C.

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