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

Mutagenesis analyses
The VP1 coding region (ORF2) of the NV was amplified and cloned into a pCI vector using the restriction sites I and I as described elsewhere . Site-directed mutagenesis of pCI-NV was performed using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA), and complementary forward and reverse primers that carried the nucleotide mutations (). The restriction enzyme I (10 U/ µl) was used to digest the parental DNA. Each of the mutated products was transformed into XL10-Gold® ultracompetent cells (Stratagene). Transformed cells were grown overnight in LB plates with carbenicillin (50 µg/ml), and individual colonies were used for plasmid amplification. The resulting plasmids were subjected to sequencing analysis to verify the entire VP1 coding region and confirm the presence of introduced mutations.

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Manufacturer protocol

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