Briefly, cDNA from plasmids purchased from Addgene was amplified by PCR using Phusion polymerase mix [New England BioLabs (NEB); M0532S] and the appropriate primers (table S4 ). PCR products were gel-purified using a QIAquick gel extraction kit (Qiagen; 28115). The constructs and the pSF-EF1α-Ub-Neo plasmid were digested with restriction enzymes from NEB (EcoR1-HF, R3101S; EcoRV-HF, R3195S; BsiW1, R0553S; BsrGI-HF, R3575S), where applicable. The plasmid was dephosphorylated with Antarctic Phosphatase (NEB; M02809S). Digested PCR products were purified with a QIAquick PCR purification kit (Qiagen, 28104) and ligated to each other and then into the pSF-EF1α-Ub-Neo plasmid using Promega LigaFast Rapid DNA Ligation kit (Fisher Scientific; PR-M8221). Portions of the ligation reactions were used to transform DH5α cells (Thermofisher; 18265017). Plasmids from single antibiotic–resistant clones were purified using Mini-prep kit(s) (Qiagen; 27104) and Maxi-prep kit(s) (Qiagen; 12362). Construction of the plasmids was verified by DNA sequencing.
Phusion polymerase mix
Manufactured by New England Biolabs
Phusion polymerase mix is a high-fidelity DNA polymerase used for amplification of DNA fragments. It exhibits increased processivity and accuracy compared to standard Taq DNA polymerases.
Automatically generated - may contain errors
Lab products found in correlation
Ligafast rapid dna ligation kit, by Thermo Fisher Scientific (2 mentions)
Miniprep kit, by Qiagen (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Maxiprep kit, by Qiagen (1 mentions)
Dh5α cells, by Thermo Fisher Scientific (1 mentions)
Antarctic phosphatase, by New England Biolabs (1 mentions)
Endofree plasmid maxi kit, by Qiagen (1 mentions)
Dh5α competent cells, by Thermo Fisher Scientific (1 mentions)
2 protocols using phusion polymerase mix
1
Plasmid Amplification and Cloning
2
Generating mEGFP-RelA Fusion Protein
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To generate the mEGFP-RelA plasmid encoding an N-terminal fusion protein, cDNA was amplified by PCR using Phusion polymerase mix (New England BioLabs (NEB); M0532S) and the indicated primers (Table S1 ). Constructs were digested with restriction enzymes from NEB (EcoR1-HF, R3101S; EcoRV-HF, R3195S; BsrGI-HF, R3575S) and ligated into the pSF-EF1α-Ub-Neo vector using Promega LigaFast Rapid DNA Ligation kit (Fisher Scientific; PR-M8221). The resulting plasmid was used to transform DH5α competent cells (ThermoFisher; 18265017). Plasmid derived and expanded from a single antibiotic resistant clone was purified using an EndoFree Plasmid Maxi kit (Qiagen; 12362). Plasmid construction was verified by DNA sequencing.
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