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

Indict

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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.