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

Reporter vector construction and site‐directed mutagenesis
AR bound enhancer fragments of AGR2 and AGR3 were PCR amplified with the primers listed in Table 2 using Phusion high fidelity DNA polymerase (Finnzyme, Espoo, Finland) and inserted into the pSC‐B entry vector (Stratagene, La Jolla, CA, US). Inserts were size verified by KpnI and SacI restriction and sequencing, followed by ligation into a PGL3 promoter plasmid (Promega, Madison, WI, USA) using the Rapid DNA Ligation Kit (Roche) to generate firefly luciferase reporter vectors. Putative AREs were deleted using the QuikChange II Site‐Directed Mutagenesis Kit (Stratagene). The mutagenic primers used were 5′‐gctgAGCACAggaCACCGAgggaatggtgc‐3′ and 5′‐ggagtcAGGACAagcCACTCCtgctgaagtag‐3′ for AGR2 S1 ARE1 (AGCACAggaTGCTGA) and ARE2 (AGGACAagcTGATCC), respectively, and 5′‐ctgggtTGAGCTttgTTTGTCagagtcaactgtccc‐3′, 5′‐ccagagtCAACTGtccGTCGCCtgtactaaaatccacc‐3′ and 5′‐cagtctttaaAGTCTCagaTGACCAaggccttaaggtac‐3′ for AGR3 S1 ARE1–3 (TGAGCTttgTGTTCC, CAACTGtccCTGTCC and AGTACAagaTGACCA).

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Manufacturer protocol

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