All plasmid expansion was performed in Stbl3 bacteria (Invitrogen). Liquid bacterial cultures were grown in a shaking bacterial incubator (New Brunswick I24) at 37°C and 225 r.p.m. Liquid bacterial cultures were grown with Luria–Bertani (LB) broth, containing the appropriate selection marker when necessary. Bacterial agar plates were cultured in a standard bacterial incubator (Labnet) at 37°C. Bacteria were cultured on LB-agar plates made by autoclaving LB with agar and pouring into bacterial dishes to solidify, with appropriate antibiotics added once the mixture cooled to 60°C. Electrocompetent Stbl3 bacteria were generated as described previously (Sambrook et al., 1989 ).
Plasmid DNA was electroporated (Bio-Rad MicroPulser) into 50 µl of electrocompetent Stbl3 bacteria (1.0×1010 cells, 0.2 cm cuvettes, Ec2 pulse setting) then recovered in 1 ml SOC medium (Howland, 1995 ) for 1 h at 37°C before plating on LB-agar plates containing the appropriate selection marker. Plasmid DNA for further subcloning was extracted from 3 ml miniprep cultures with QIAprep Spin Miniprep Kit (Qiagen 27106). Plasmid DNA for transfection into mammalian cells was extracted from 100 ml midiprep cultures with the NucleoBond Xtra Midi Plus EF kit (Macherey-Nagel). Details of reagents can be found inTable S5.
Plasmid DNA was electroporated (Bio-Rad MicroPulser) into 50 µl of electrocompetent Stbl3 bacteria (1.0×1010 cells, 0.2 cm cuvettes, Ec2 pulse setting) then recovered in 1 ml SOC medium (Howland, 1995 ) for 1 h at 37°C before plating on LB-agar plates containing the appropriate selection marker. Plasmid DNA for further subcloning was extracted from 3 ml miniprep cultures with QIAprep Spin Miniprep Kit (Qiagen 27106). Plasmid DNA for transfection into mammalian cells was extracted from 100 ml midiprep cultures with the NucleoBond Xtra Midi Plus EF kit (Macherey-Nagel). Details of reagents can be found in