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

In vitro mutagenesis of Rab7 and Rab11 mutant plasmids.
To generate mutant Rab7 and Rab11 plasmids expressing CFP-Rab7Q70L, CFP-Rab7T22N, GFP-Rab11Q70L, and GFP-Rab11S25N (30, 31), we performed in vitro mutagenesis using a QuikChange site-directed mutagenesis kit. The wild-type plasmids CFP-Rab7 and GFP-Rab11 were used as the templates. Mutagenesis was performed using the following primer pairs: for CFP-Rab7Q70L, 5′-ATATGGGACACAGCAGGACTGGAACGGTTCCAGTCTCTC-3′ and 5′-GAGAGACTGGAACCGTTCCAGTCCTGCTGTGTCCCATAT-3′; for CFP-Rab7T22N, 5′-GATTCTGGAGTCGGGAAGAACTCACTCATGAACCAGTAT-3′ and 5′-ATACTGGTTCATGAGTGAGTTCTTCCCGACTCCAGAATC-3′; for GFP-Rab11Q70L, 5′-ATATGGGACACAGCAGGGCTAGAGCGATATCGAGCTATA-3′ and 5′-TATAGCTCGATATCGCTCTAGCCCTGCTGTGTCCCATAT-3′; and for GFP-Rab11S25N, 5′-GATTCTGGTGTTGGAAAGAATAATCTCCTGTCTCGATTT-3′ and 5′-AAATCGAGACAGGAGATTATTCTTTCCAACACCAGAATC-3′. The sequences of the mutant DNA fragments were confirmed by DNA sequencing.

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Manufacturer protocol

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