X-tremeGENE™ HP DNA Transfection Reagent

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Do not aliquot X-tremeGENE HP DNA Transfection Reagent; store in the original glass vials.

Protocol tips
Do not aliquot X-tremeGENE HP DNA Transfection Reagent; store in the original glass vials.

Publication protocol

Site-directed mutagenesis and gene overexpression of kinesin-12 mutants
The QuikChange® Lightning Site-Directed Mutagenesis Kit (Stratagene) was used to generate the mutant kinesin-12-T1142A (phospho-mutant) and kinesin-12-T1142D (phospho-mimic) constructs. The mutagenesis primers (listed in Table S1) design and performance procedures were according to the manufacturer's instructions, and all the constructs were confirmed by DNA sequencing (BGI, Shanghai). The GFP empty vector, GFP–kinesin-12 wild-type, GFP–kinesin-12-T1142D or GFP–kinesin-12-T1142A constructs were transfected into cultures astrocytes using X-tremeGENE HP DNA Transfection Reagent following the manufacturer's instructions. After expression for the scheduled time, the cells were dissociated for cell sorting, depending on the GFP fluorescence channel, using Flow cytometery (FACS-Aria, BD). GFP-positive cells were identified using a 488-nm laser. The obtained cells were used for the Transwell migration assay, for colocalization analysis by immunostaining, or in the co-immunoprecipitation assay as above.

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Manufacturer protocol

Download the product protocol from Sigma-Aldrich for X-tremeGENE™ HP DNA Transfection Reagent below.

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