PvuI (10 U/µL)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	An expression plasmid coding for the full-length XPG/XPF protein or one of the splice variants, e.g. pcDNA3.1 (+)XPF, was linearized using the PvuI restriction enzyme (Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer’s instructions.	Successful linearization was analyzed on an agarose gel and the linearized plasmids were purified by ethanol precipitation.

Protocol tips
An expression plasmid coding for the full-length XPG/XPF protein or one of the splice variants, e.g. pcDNA3.1 (+)XPF, was linearized using the PvuI restriction enzyme (Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer’s instructions.
Downstream tips
Successful linearization was analyzed on an agarose gel and the linearized plasmids were purified by ethanol precipitation.

Publication protocol

Stable cell lines overexpressing the full-length protein or respective splice variants were generated via spontaneous integration of a linearized expression plasmid into MRC5Vi WT cells. An expression plasmid coding for the full-length XPG/XPF protein or one of the splice variants, e.g. pcDNA3.1 (+)XPF, was linearized using the PvuI restriction enzyme (Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer’s instructions. Successful linearization was analyzed on an agarose gel and the linearized plasmids were purified by ethanol precipitation. Afterwards, the linearized plasmids were transiently transfected into MRC5Vi WT cells seeded on 10mm cell culture dishes and positive clones were selected using G418. Single cell clones were established by serial dilution and checked for plasmid integration via PCR using specific primer pairs for the CMV (cytomegalo virus)-promoter and the integrated gene (see Table ​Table1),1), as well as immunoblot analyses.

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Manufacturer protocol

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