XmaJI (AvrII) (10 U/µL)

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	The resulting PCR product was digested with XmaJI and DpnI (Thermo Scientific) to cleave residual methylated template plasmid and ligated using T4 ligase (Thermo Scientific).

Protocol tips
The resulting PCR product was digested with XmaJI and DpnI (Thermo Scientific) to cleave residual methylated template plasmid and ligated using T4 ligase (Thermo Scientific).

Publication protocol

"The rocAM1 sequence was amplified by PCR from genomic DNA extracted from strain 854 and cloned into the vector pDL278 as described previously (26). A FLAG tag was incorporated immediately upstream of the rocA stop codon by overlap PCR using primers pDLrocAFLAG_F and _R (Table 2), which incorporate XmaJI sites into both ends of the PCR product. The resulting product was digested with XmaJI and DpnI (Thermo Scientific) to cleave residual methylated template plasmid and ligated using T4 ligase (Thermo Scientific). The resulting construct was designated pDL_rocAM1FLAG.The rocAM3 sequence was amplified from genomic DNA extracted from strain 188 using primers pDL_rocAM3_F and _R (Table 2) and cloned into the vector pDL278 using restriction enzyme BamHI (Thermo Scientific) and T4 ligase (Thermo Scientific). The resulting construct was designated pDL_rocAM3FLAG.

Plasmid pDL_rocATMD_FLAG, expressing the N-terminal RocA TMDs, was generated from plasmid pDLrocAM1_FLAG by overlap PCR. The entire plasmid was amplified using primers pDL_rocATMD_FLAG_F and _R (Table 2) to incorporate a stop codon at amino acid 226 and XmaJI restriction sites at both ends of the amplicon. The resulting PCR product was digested with XmaJI and DpnI (Thermo Scientific) to cleave residual methylated template plasmid and ligated using T4 ligase (Thermo Scientific).

Plasmid pDL_rocACYT_FLAG, expressing the C-terminal RocA cytoplasmic domain was generated from plasmid pDLrocAM1_FLAG by overlap PCR. Primers pDL_rocACYT_FLAG_F and _R (Table 2) were used to amplify pDLrocAM1_FLAG and remove amino acids 1 to 225 of the rocA gene product from the resulting PCR product. NdeI restriction sites were incorporated into both ends of the amplicon, providing a new, in-frame start codon for expression of RocACYT_FLAG in vivo. The resulting PCR product was digested with NdeI and DpnI (Thermo Scientific) to cleave residual methylated template plasmid and ligated using T4 ligase (Thermo Scientific).

All resulting plasmids were transformed into GAS strain H566 by electroporation as described previously (26)."

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