QuikChange Site-Directed Mutagenesis Kit, 10 Rxn

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- XL10-Gold cells are resistant to tetracycline and chloramphenicol. If the mutagenized plasmid contains only the tetR or camR resistance marker, an alternative strain of competent cells must be used.	- Use only the Dpn I enzyme provided; do not substitute with an enzyme from another source.

Upstream tips
- XL10-Gold cells are resistant to tetracycline and chloramphenicol. If the mutagenized plasmid contains only the tetR or camR resistance marker, an alternative strain of competent cells must be used.
Protocol tips
- Use only the Dpn I enzyme provided; do not substitute with an enzyme from another source.

Publication protocol

Plasmid constructs
A pOTB7 plasmid encoding wild-type human CD44 (imaGenes; Source Bioscience) was grown on Luria-Bertani agar plates with 20 μg/ml chloramphenicol and subsequently in Luria-Bertani broth with the same antibiotic concentration. Plasmid extraction was carried out using Qiagen Midi-Prep kits following the manufacturer’s protocols (Qiagen, Manchester, UK). This preparation was used as template genetic material for site-directed mutagenesis of CD44 palmitoylation sites. Single point mutations were obtained using the QuikChange Site-Directed Mutagenesis kit (Agilent Technologies Ireland Ltd, Cork, Ireland). Cys286 was mutated to either serine (C286S) or alanine (C286A). A double mutation was achieved using the mutated Cys286 DNA as a template for introducing a mutation at Cys295 to alanine. The mutagenesis primers were obtained from Eurofins MWG Operon (Ebersberg, Germany) and contained the sequences presented in Table 1. All mutations were confirmed by sequencing of the synthesised plasmids (Source Bioscience) and alignment using BLAST software (National Institute of Health, Bethesda, MD, USA). It is noteworthy that mammalian expression of pOTB7 plasmid inserts is normally achieved by subcloning via the Gateway expression system (Life Technologies, Paisley, UK). However, based on the precedent that mammalian expression can be successfully achieved without the subcloning step [20] (albeit via an unknown mechanism), we did not use the Gateway step.

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Manufacturer protocol

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