Titlebook: In Vitro Mutagenesis Protocols; Michael K. Trower Book 19961st edition Humana Press 1996

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Site-Directed Mutagenesis In Vitro by Megaprimer PCR,uct (A-M1) is purified and used as one of the primers (hence the name “megaprimer”) in the second round of PCR along with the other flanking primer (B). The wild-type cloned gene is used as template in both PCR reactions. The final PCR product (A-M1-B) containing the mutation can be used in a variet

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Li Zhuevy del Aguila Marchena. is Senior Professor and Chair of the Department of Management Sciences at the Pontificia Universidad Católica del Perú. He has published extensively on Marx, political philosophy, and applied ethics..978-3-030-82896-7978-3-030-82894-3Series ISSN 2524-7123 Series E-ISSN 2524-7131

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In Vitro Site-Directed Mutagenesis Using the Unique Restriction Site Elimination (USE) Method,parental (wild-type) plasmid. The USE strategy employs two oligonucleotide primers: one primer (the mutagenic primer) produces the desired mutation, whereas the second primer (the selection primer) mutates a restriction site unique to the plasmid for the purpose of selection.

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Site-Directed Mutagenesis Using a Uracil-Containing Phagemid Template, given DNA sequence with high efficiency. I have used this procedure successfully many times and most recently to map protein interaction sites within the activation domain of the transcription factor E2F (.).

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A Simple Method for Site-Directed Mutagenesis with Double-Stranded Plasmid DNA,. Limitations of these methods include the availability of restriction sites for subcloning and the instability of large inserts in M13 vectors (.), the low fidelity of . polymerase, the cost of multiple primers in the PCR protocols, and the low mutant yields with the double-stranded plasmid method.

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Ordered Deletions Using Exonuclease III,.). ExoIII digests one strand of double-stranded DNA by removing nucleotides from 3′ ends if the end is blunt or has a 5′ protrusion. A 3′ protrusion of 4 bases or more is resistant to ExoIII digestion.

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PCR-Based Site-Directed Mutagenesis, mutagenesis reaction (.). By using this method, a series of site-directed mutations may be undertaken that only require a single primer for each desired change, and furthermore, no reiterative transformation steps are necessary.