AatII NEB#R0117

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Following isolation from E. coli strain Top10 using a Plasmid Midi Kit (Qiagen, Valencia, CA) according to manufacturer protocols, plasmid pXG-10 [54] was digested with AatII and NheI restriction endonucleases (New England BioLabs, Ipswich, MA) to remove the PLtetO-1 promoter and lacZ fragment. A DNA fragment containing the shuA start codon and 390 upstream nucleotides was amplified from the chromosome of wild-type S. dysenteriae by polymerase chain reaction using primers which contain AatII and NheI endonuclease recognition sites respectively. The amplified product was purified using a QIAQuick gel extraction kit (Qiagen) and then digested with AatII and NheI endonucleases and cloned into the digested pXG-10 plasmid backbone to create pshuA-gfp. The nucleic acid sequence of pshuA-gfp was verified by nucleic acid sequencing of both DNA strands.

Protocol tips

Following isolation from E. coli strain Top10 using a Plasmid Midi Kit (Qiagen, Valencia, CA) according to manufacturer protocols, plasmid pXG-10 [54] was digested with AatII and NheI restriction endonucleases (New England BioLabs, Ipswich, MA) to remove the PLtetO-1 promoter and lacZ fragment. A DNA fragment containing the shuA start codon and 390 upstream nucleotides was amplified from the chromosome of wild-type S. dysenteriae by polymerase chain reaction using primers which contain AatII and NheI endonuclease recognition sites respectively. The amplified product was purified using a QIAQuick gel extraction kit (Qiagen) and then digested with AatII and NheI endonucleases and cloned into the digested pXG-10 plasmid backbone to create pshuA-gfp. The nucleic acid sequence of pshuA-gfp was verified by nucleic acid sequencing of both DNA strands.

Publication protocol

Following isolation from E. coli strain Top10 using a Plasmid Midi Kit (Qiagen, Valencia, CA) according to manufacturer protocols, plasmid pXG-10 [54] was digested with AatII and NheI restriction endonucleases (New England BioLabs, Ipswich, MA) to remove the PLtetO-1 promoter and lacZ fragment. A DNA fragment containing the shuA start codon and 390 upstream nucleotides was amplified from the chromosome of wild-type S. dysenteriae by polymerase chain reaction using primers which contain AatII and NheI endonuclease recognition sites respectively. The amplified product was purified using a QIAQuick gel extraction kit (Qiagen) and then digested with AatII and NheI endonucleases and cloned into the digested pXG-10 plasmid backbone to create pshuA-gfp. The nucleic acid sequence of pshuA-gfp was verified by nucleic acid sequencing of both DNA strands.

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