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

3′UTR luciferase reporter assay
A fragment of 3′UTR of Med1 or Med31 containing the putative miR-1 binding site (420–427 or 1059–1069, respectively) was amplified by PCR using the following primers: wt-Med1 (forward), 5′-GAA AGGCATATCCAGACCCTATT-3′ and wt-Med1 (reverse), 5′-GGAAGGCTGTCCTACACTAAAC-3′; wt-Med31 (forward), 5′-TCTCCTGGAACCTTACTGTCT-3′ and wt-Med31 (reverse), 5′-GCAACTGATGATATTCCTGA AACC-3′. The PCR product was subcloned into a pMIR-REPORT vector (Ambion) to generate the pMIR-Report-Med1/Med31 wt plasmid. Site-directed mutagenesis of miR-1 binding sites was carried out using a QuikChange site-directed mutagenesis kit (Stratagene, Santa Clara, CA, USA). All constructs were verified by DNA sequencing.

For reporter assays, U2OS cells were plated in 96-well clusters, then cotransfected with 0.3 μg wt or mutant reporter plasmids and 60 nM miR-1 precursors. At 48 h after transfection, luciferase activity was measured and normalized by the control vector containing Renilla luciferase.

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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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