Phusion Site-Directed Mutagenesis Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Phusion Hot Start II DNA Polymerase tends to work better at elevated denaturation and annealing temperatures due to higher salt concentrations in its buffer. Hence follow manufacturer instruction strictly.

Upstream tips
Phusion Hot Start II DNA Polymerase tends to work better at elevated denaturation and annealing temperatures due to higher salt concentrations in its buffer. Hence follow manufacturer instruction strictly.

Publication protocol

Cloning and site-directed mutagenesis of the SGLT1 promoter
For promoter assays, the previously characterized human SGLT1 promoter (−1968/+14, designated WT SGLT1-Luc) [21] was amplified by PCR from human genomic DNA (Promega, Madison, WI) using primers encoding NotI and NheI restriction sites at the 5′ and 3′ ends respectively (Supplemental Table 3) and inserted into the pcDNA3.1(+) vector (Invitrogen). To test the function of E-box sites within the SGLT1 promoter, site-directed mutagenesis was performed on the pGL3-SGLT1 vector created above using the Phusion Site-directed Mutagenesis kit (NEB, Ipswich, MA). E-boxes were mutated by conversion to NheI restriction sites (GCTAGC) and designated SGLT1-Luc mutEB1 through mutEB4. Single E-box mutations were introduced using a forward primer that incorporated the NheI restriction site in place of the E-box sequence and 15–20 complementary bases on either side and a fully complementary reverse primer. Site-directed mutagenesis primers were acquired from Invitrogen (Supplemental Table 3). To assess synergistic interactions, combinations of E-box mutations (designated mutEB1+2, 1+3 and 1+2+3) were prepared by sequential site-directed mutagenesis of mutated promoters. All constructs were sequenced to confirm successful mutation.

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for Phusion Site-Directed Mutagenesis Kit below.

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