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

Vector construction and luciferase reporter assay
A human SIRT1 3′UTR was amplified from renal cancer cell line cDNAs and cloned into the XhoI/NotI site of a psiCHECK-2 vector (Promega, Madison, WI, USA). For mutagenesis of the miR-22-binding site, a QuikChange Site-Directed Mutagenesis kit (Agilent Technologies, Palo Alto, CA, USA) was used following the manufacturer's instructions.

For luciferase activity assay, 786-O cells were seeded into 12-well plates overnight before transfection, and then co-transfected with 200 ng of psiCHECK-2 vectors, which harbored SIRT1 3′UTR wild-type or mutant constructs, and 200 nM of miR-22 or miR-NC. Forty-eight hours after transfection, luciferase activity was measured with a Dual-Luciferase assay kit (Promega) according to the manufacturer's instructions. Renilla luciferase activity was normalized to firefly luciferase activity.

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Manufacturer protocol

Download the product protocol from Agilent Technologies for QuikChange Site-Directed Mutagenesis Kit, 10 Rxn below.

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