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Phusion site directed mutagenesis kit protocol

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The Phusion site-directed mutagenesis kit protocol is a laboratory tool used to introduce specific mutations into DNA sequences. It provides a straightforward method for making targeted changes to plasmid or other DNA constructs.

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Lab products found in correlation

Quikchange site directed mutagenesis kit protocol, by Agilent Technologies (2 mentions)

Close spelling variants of this product

Phusion site-directed mutagenesis protocol Phusion Site-Directed Mutagenesis Site-directed mutagenesis Phusion Mutagenesis kit Phusion mutagenesis

2 protocols using phusion site directed mutagenesis kit protocol

1

Screening KEAP1 Mutants for CBR-470-1 Response

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KEAP1 mutants were generated with PCR primers in Extended Data Table 1 according to the Phusion site-directed mutagenesis kit protocol (F-541, Thermo Scientific) and the QuikChange site-directed mutagenesis kit protocol (200523, Agilent). Mutant KEAP1 plasmids were verified by sequencing [CMV (forward), wild-type primers in the middle of KEAP1 sequence (forward) and BGH (reverse)], and were transiently expressed in HEK293T cells in the same manner as wild type KEAP1. Screening of CBR-470-1-induced HMW-KEAP1 formation with mutant constructs was performed just as with wild type KEAP1, after 8 hour CBR-470-1 treatment (20 μM). Following treatment, cells were harvested and prepared for SDS-PAGE and western blotting as indicated above.
Bollong M.J., Lee G., Coukos J.S., Yun H., Zambaldo C., Chang J.W., Chin E.N., Ahmad I., Chatterjee A.K., Lairson L.L., Schultz P.G, & Moellering R.E. (2018). A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling. Nature, 562(7728), 600-604.
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2

Screening KEAP1 Mutants for CBR-470-1 Response

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KEAP1 mutants were generated with PCR primers in Extended Data Table 1 according to the Phusion site-directed mutagenesis kit protocol (F-541, Thermo Scientific) and the QuikChange site-directed mutagenesis kit protocol (200523, Agilent). Mutant KEAP1 plasmids were verified by sequencing [CMV (forward), wild-type primers in the middle of KEAP1 sequence (forward) and BGH (reverse)], and were transiently expressed in HEK293T cells in the same manner as wild type KEAP1. Screening of CBR-470-1-induced HMW-KEAP1 formation with mutant constructs was performed just as with wild type KEAP1, after 8 hour CBR-470-1 treatment (20 μM). Following treatment, cells were harvested and prepared for SDS-PAGE and western blotting as indicated above.
Bollong M.J., Lee G., Coukos J.S., Yun H., Zambaldo C., Chang J.W., Chin E.N., Ahmad I., Chatterjee A.K., Lairson L.L., Schultz P.G, & Moellering R.E. (2018). A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signaling. Nature, 562(7728), 600-604.
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