QuikChange Multi Site-Directed Mutagenesis Kit

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

Reporter gene assays
LNCaP cells were seeded in 24-well plates at a density of 70 000 cells/well phenol red-free RPMI medium supplemented with 10% DSS. Forty-eight hours later, the cell medium was replaced with fresh RPMI phenol red-free medium supplemented with 10% DSS with or without 1 nM R1881. Each well was transfected with 500 ng DNA, consisting of: 100 ng of firefly luciferase pGL3 vectors, 100 ng luciferase vector (pRL-TK) and 300 ng Bluescribe plasmid DNA as carrier DNA. Cells were transfected using Lipofectamine LTX and PLUS Reagent (Invitrogen) according to the manufacturer's instructions. Twenty-four hours following transfection, firefly and luciferase activities were determined using the Dual-Glo Luciferase Assay System (Promega). Firefly luciferase readings were subsequently normalized against the control luciferase such that the luciferase activity served to control for transfection efficiency.

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Manufacturer protocol

Download the product protocol from Agilent Technologies for QuikChange Multi Site-Directed Mutagenesis Kit below.

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