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.

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

Publication protocol

Isolation of OCLN-Knockout Cells Using the CRISPR/
Cas9 System Huh7.5.1-8 cells were seeded at a density of
5×104
cells/well in a 48-well plate and incubated overnight.
Cells were transfected with 0.3 µg of each plasmid (pX330-site
A and pX330-site B) using 1.2 µL of the X-tremeGENE HP
DNA Transfection Reagent (Roche Diagnostics, Mannheim,
Germany), according to the manufacturer’s instruction. Transfections were performed twice 48 h apart. Transfected cells
were infected with HCV-JFH1 at a multiplicity of infection
(MOI) of 10 and cultured for 9 d. HCV-resistant cells were
cloned by limiting dilution. One of the established clones
was named OKH-4 (OCLN-knockout Huh7.5.1-8-derived
cell clone 4). If OCLN is essential for HCV entry, OCLNknockout cells would be resistant to HCV and the selection of HCV-resistant cells is efficient to concentrate OCLN-knockout
cells. That was the case. All selected and cloned cells were
OCLN-knockout cells (see Results).

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Manufacturer protocol

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

Download manufacturer protocol

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