The Experts below are selected from a list of 324 Experts worldwide ranked by ideXlab platform
Ruth M Ruprecht - One of the best experts on this subject based on the ideXlab platform.
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One-step, highly efficient Site-Directed Mutagenesis by toxic protein selection
BioTechniques, 2002Co-Authors: Weidong Xu, Claudia R. Ruprecht, Thipparthi R. Reddy, B. A. Mcfadden, Flossie Wong-staal, Yingying Zhang, Ruth M RuprechtAbstract:A fast and efficient Site-Directed Mutagenesis method has been developed, using the newly constructed plasmid pTPS19, which expresses the toxic CcdB protein originally encoded by the E. coli F plas...
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One-step, highly efficient Site-Directed Mutagenesis by toxic protein selection
BioTechniques, 2002Co-Authors: Weidong Xu, Claudia R. Ruprecht, Thipparthi R. Reddy, B. A. Mcfadden, Flossie Wong-staal, Yingying Zhang, Ruth M RuprechtAbstract:A fast and efficient Site-Directed Mutagenesis method has been developed, using the newly constructed plasmid pTPS19, which expresses the toxic CcdB protein originally encoded by the E. coli F plas...
Luying Xun - One of the best experts on this subject based on the ideXlab platform.
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Revised Mechanism and Improved Efficiency of the QuikChange Site-Directed Mutagenesis Method
Methods in molecular biology (Clifton N.J.), 2016Co-Authors: Yongzhen Xia, Luying XunAbstract:Site-Directed Mutagenesis has been widely used for the substitution, addition or deletion of nucleotide residues in a defined DNA sequence. QuikChange™ Site-Directed Mutagenesis and its related protocols have been widely used for this purpose because of convenience and efficiency. We have recently demonstrated that the mechanism of the QuikChange™ Site-Directed Mutagenesis process is different from that being proposed. The new mechanism promotes the use of partially overlapping primers and commercial PCR enzymes for efficient PCR and Mutagenesis.
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new insights into the quikchangetm process guide the use of phusion dna polymerase for Site Directed Mutagenesis
Nucleic Acids Research, 2015Co-Authors: Yongzhen Xia, Wenqiao Chu, Luying XunAbstract:The QuikChange™ Site-Directed Mutagenesis method is popular but imperfect. An improvement by using partially overlapping primers has been reported several times; however, it is incompatible with the proposed mechanism. The QuikChange™ method using complementary primers is proposed to linearly amplify a target plasmid with the products annealing to produce double-stranded DNA molecules with 5'-overhangs. The overhang annealing is supposed to form circular plasmids with staggered breaks, which can be repaired in Escherichia coli after transformation. Here, we demonstrated that the PCR enzyme fills the 5'-overhangs in the early cycles, and the product is then used as the template for exponential amplification. The linear DNA molecules with homologous ends are joined to generate the plasmid with the desired mutations through homologous recombination in E. coli. The correct understanding is important to method improvements, guiding us to use partially overlapping primers and Phusion DNA polymerase for Site-Directed Mutagenesis. Phusion did not amplify a plasmid with complementary primers but used partially overlapping primers to amplify the plasmid, producing linear DNA molecules with homologous ends for Site-Directed Mutagenesis.
Weidong Xu - One of the best experts on this subject based on the ideXlab platform.
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One-step, highly efficient Site-Directed Mutagenesis by toxic protein selection
BioTechniques, 2002Co-Authors: Weidong Xu, Claudia R. Ruprecht, Thipparthi R. Reddy, B. A. Mcfadden, Flossie Wong-staal, Yingying Zhang, Ruth M RuprechtAbstract:A fast and efficient Site-Directed Mutagenesis method has been developed, using the newly constructed plasmid pTPS19, which expresses the toxic CcdB protein originally encoded by the E. coli F plas...
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One-step, highly efficient Site-Directed Mutagenesis by toxic protein selection
BioTechniques, 2002Co-Authors: Weidong Xu, Claudia R. Ruprecht, Thipparthi R. Reddy, B. A. Mcfadden, Flossie Wong-staal, Yingying Zhang, Ruth M RuprechtAbstract:A fast and efficient Site-Directed Mutagenesis method has been developed, using the newly constructed plasmid pTPS19, which expresses the toxic CcdB protein originally encoded by the E. coli F plas...
Claudia R. Ruprecht - One of the best experts on this subject based on the ideXlab platform.
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One-step, highly efficient Site-Directed Mutagenesis by toxic protein selection
BioTechniques, 2002Co-Authors: Weidong Xu, Claudia R. Ruprecht, Thipparthi R. Reddy, B. A. Mcfadden, Flossie Wong-staal, Yingying Zhang, Ruth M RuprechtAbstract:A fast and efficient Site-Directed Mutagenesis method has been developed, using the newly constructed plasmid pTPS19, which expresses the toxic CcdB protein originally encoded by the E. coli F plas...
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One-step, highly efficient Site-Directed Mutagenesis by toxic protein selection
BioTechniques, 2002Co-Authors: Weidong Xu, Claudia R. Ruprecht, Thipparthi R. Reddy, B. A. Mcfadden, Flossie Wong-staal, Yingying Zhang, Ruth M RuprechtAbstract:A fast and efficient Site-Directed Mutagenesis method has been developed, using the newly constructed plasmid pTPS19, which expresses the toxic CcdB protein originally encoded by the E. coli F plas...
Thipparthi R. Reddy - One of the best experts on this subject based on the ideXlab platform.
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One-step, highly efficient Site-Directed Mutagenesis by toxic protein selection
BioTechniques, 2002Co-Authors: Weidong Xu, Claudia R. Ruprecht, Thipparthi R. Reddy, B. A. Mcfadden, Flossie Wong-staal, Yingying Zhang, Ruth M RuprechtAbstract:A fast and efficient Site-Directed Mutagenesis method has been developed, using the newly constructed plasmid pTPS19, which expresses the toxic CcdB protein originally encoded by the E. coli F plas...
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One-step, highly efficient Site-Directed Mutagenesis by toxic protein selection
BioTechniques, 2002Co-Authors: Weidong Xu, Claudia R. Ruprecht, Thipparthi R. Reddy, B. A. Mcfadden, Flossie Wong-staal, Yingying Zhang, Ruth M RuprechtAbstract:A fast and efficient Site-Directed Mutagenesis method has been developed, using the newly constructed plasmid pTPS19, which expresses the toxic CcdB protein originally encoded by the E. coli F plas...
