Q5® Site-Directed Mutagenesis Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- Only use 1 μl of PCR product in the KLD reaction. Carrying too much PCR product forward can decrease transformation efficiency. - Only use 5 μl of the KLD reaction in the transformation. If more KLD reaction is added, a buffer exchange step, such as PCR purification, should be included prior to transformation.

Protocol tips
- Only use 1 μl of PCR product in the KLD reaction. Carrying too much PCR product forward can decrease transformation efficiency. - Only use 5 μl of the KLD reaction in the transformation. If more KLD reaction is added, a buffer exchange step, such as PCR purification, should be included prior to transformation.

Publication protocol

Site-directed Mutagenesis
The cDNA encoding wild type (wt) CLN5 was purchased from GeneCopoeia and cloned into pcDNA3.1/Myc-His(−)A using EcoRI and BamHI restriction sites. To generate individual N-glycosylation mutants, the codon for Asn in the consensus sequence for N-glycosylation was mutated to a codon for Gln using phusion-based site-directed mutagenesis (NEB). The cDNAs containing the single mutations for the N-glycosylation sites served as templates for creating multiple N-glycosylation mutants. All constructs were confirmed by sequence analysis.

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Manufacturer protocol

Download the product protocol from New England BioLabs for Q5® Site-Directed Mutagenesis Kit below.

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