Amicon ultracel 0.5ml spin concentrator

In vitro synthesis of the RmuC domains of ShdA I, ShdA II, ShdA III and ShdA IV was performed using the PURExpress cell-free transcription/translation kit (NEB). Protein synthesis was performed with either 250 ng and 500 ng of DNA template. Synthesis was performed for 4 hr at 37°C according to the manufacturer’s recommendation. Following incubation, 10 mM MgCl₂ was added to the reaction and the final volume was adjusted to 10 μL. Ribosomes were removed through centrifugation for 60 min at 15,000 rpm at 4°C, through an Amicon Ultracel 0.5mL spin concentrator with a 100 KDa filter (Merck). The flowthrough was collected and the His-tagged PURExpress kit components were removed from the reaction following incubation with Ni-NTA agarose beads (Thermo) for 45 min at 4°C. Agarose beads were removed through centrifugation with Biorad micro Bio-spin columns at 15,000 g for 10 min at 4°C. As a control, the same reactions were performed in parallel with the dihydrofolate reductase (DHFR) control provided by the PURExpress kit.
To test for nuclease activity the in vitro synthesised ShdA proteins or DHFR control were incubated with 20 ng of phage ϕSipho, E. coli MG1655 chromosome or plasmid pSG483 DNA, followed by agarose gel electrophoresis and staining with GelRed (Cambridge Bioscience).

In vitro synthesis of the RmuC domains of ShdA I, ShdA II, ShdA III and ShdA IV was performed using the PURExpress cell-free transcription/translation kit (NEB). Protein synthesis was performed with either 250 ng and 500 ng of DNA template. Synthesis was performed for 4 hr at 37°C according to the manufacturer’s recommendation. Following incubation, 10 mM MgCl₂ was added to the reaction and the final volume was adjusted to 10 μL. Ribosomes were removed through centrifugation for 60 min at 15,000 rpm at 4°C, through an Amicon Ultracel 0.5mL spin concentrator with a 100 KDa filter (Merck). The flowthrough was collected and the His-tagged PURExpress kit components were removed from the reaction following incubation with Ni-NTA agarose beads (Thermo) for 45 min at 4°C. Agarose beads were removed through centrifugation with Biorad micro Bio-spin columns at 15,000 g for 10 min at 4°C. As a control, the same reactions were performed in parallel with the dihydrofolate reductase (DHFR) control provided by the PURExpress kit.
To test for nuclease activity the in vitro synthesised ShdA proteins or DHFR control were incubated with 20 ng of phage ϕSipho, E. coli MG1655 chromosome or plasmid pSG483 DNA, followed by agarose gel electrophoresis and staining with GelRed (Cambridge Bioscience).