QuikChange II XL Site-Directed Mutagenesis Kit, 10 Rxn

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- Use only the Dpn I enzyme provided; do not substitute with an enzyme from another source.

Protocol tips
- Use only the Dpn I enzyme provided; do not substitute with an enzyme from another source.

Publication protocol

2.5. Site-directed mutagenesis, transient transfection, and luciferase assays of the AhR reporter construct
The AHR promoter-luciferase reporter plasmid, pGL3-hAhRP [20], which incorporates an AHR promoter insert that encompasses 5640 bp of the AHR 5′-flanking region, was used, and a deletion construct of this vector was prepared by restriction endonuclease digestion with KpnI (Gibco, Life Technologies) and ApaI (Fermentas, Thermo Fisher), yielding a construct encompassing 120 bp of the proximal AHR promoter region as described [20]. This deletion construct is referred to as AhRΔ(−120). The AHR promoter contains a GC-rich region directly upstream of the transcriptional start site (TSS). In the pGL3-hAhRP plasmid, this GC-rich region contained (GGGGC)4, which represents two additional Sp1 consensus binding sites in comparison with the Ensembl sequence for the AHR gene, which contains (GGGGC)2, found at -37 bp upstream of the TSS [21]. Since one report [22] indicated that the (GGGGC)2 was the most prominent allele, two repeats, or 10 bp, were removed from the AhRΔ(−120) deletion construct using the QuikChange II Site-Directed Mutagenesis Kit (Stratagene, Agilent Technologies) according to the manufacturer’s instructions, followed by introduction of point mutations in putative transcription factor binding sites using site-directed mutagenesis. Briefly, complementary forward and reverse primers were designed to introduce point mutations within the core sequence of the transcription factor binding sequence of interest in the AHR promoter, which were flanked by unaltered DNA sequences (Supplementary Table 1). These primers were included in a PCR reaction containing 10 ng of vector dsDNA and 2.5 U PfuUltra HF DNA polymerase (Stratagene, Agilent Technologies). PCR conditions are outlined in Supplementary Table 2. Template DNA was digested with 10 U DpnI, and cDNA plasmids containing the mutation were transformed into XL1-Blue supercompetent cells (Stratagene, Agilent Technologies). Colonies were selected with ampicillin, endonuclease-free DNA was prepared from each colony (PureYield Plasmid Midiprep System, Promega, Madison, WI), and regions of the AHR promoter were sequenced. AHR promoter sequences containing the target mutation were then subcloned into the original pGL3-basic vector (Promega).

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Manufacturer protocol

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

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