QuikChange Site-Directed Mutagenesis Kit, 10 Rxn

Site Directed Mutagenesis (SDM) Human - Point mutation U2OS Med31

Experiment
Site Directed Mutagenesis (SDM) Human - Point mutation U2OS Med31
Product
QuikChange Site-Directed Mutagenesis Kit, 10 Rxn from Agilent Technologies
Manufacturer
Agilent Technologies

Protocol tips

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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Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Site Directed Mutagenesis (SDM) Human - Point mutation U2OS Med31 using QuikChange Site-Directed Mutagenesis Kit, 10 Rxn from Agilent Technologies.

Paper title
MicroRNA-1 functions as a potential tumor suppressor in osteosarcoma by targeting Med1 and Med31.
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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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