Q5® Site-Directed Mutagenesis Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- Only use 1 μl of PCR product in the KLD reaction. Carrying too much PCR product forward can decrease transformation efficiency. - Only use 5 μl of the KLD reaction in the transformation. If more KLD reaction is added, a buffer exchange step, such as PCR purification, should be included prior to transformation.

Protocol tips
- Only use 1 μl of PCR product in the KLD reaction. Carrying too much PCR product forward can decrease transformation efficiency. - Only use 5 μl of the KLD reaction in the transformation. If more KLD reaction is added, a buffer exchange step, such as PCR purification, should be included prior to transformation.

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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Papers

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Manufacturer protocol

Download the product protocol from New England BioLabs for Q5® Site-Directed Mutagenesis Kit below.

Download manufacturer protocol

Videos

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