High-efficiency counterselection recombineering for site-directed mutagenesis in bacterial artificial chromosomes

2011 ◽  
Vol 9 (1) ◽  
pp. 103-109 ◽  
Author(s):  
Alexander W Bird ◽  
Axel Erler ◽  
Jun Fu ◽  
Jean-Karim Hériché ◽  
Marcello Maresca ◽  
...  
Keyword(s):  
High Efficiency ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Bacterial Artificial Chromosomes ◽  
Artificial Chromosomes

Multiplex site-directed mutagenesis strategy including high-efficiency selection of the mutant PCR products

2007 ◽  
Vol 29 (12) ◽  
pp. 1921-1925 ◽  
Author(s):  
Zoltán Varga-Orvos ◽  
Zsolt B. Nagy ◽  
Attila Mészáros ◽  
Szabolcs Kökény ◽  
Péter Gergely ◽  
...  
Keyword(s):  
High Efficiency ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Pcr Products ◽  
Selection Of

scholarly journals A high-efficiency method for site-directed mutagenesis of large plasmids based on large DNA fragment amplification and recombinational ligation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kewei Zhang ◽  
Xiaomei Yin ◽  
Kaituo Shi ◽  
Shihua Zhang ◽  
Juan Wang ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
High Efficiency ◽  
Dna Amplification ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Filamin A ◽  
Large Plasmids ◽  
Dna Fragment ◽  
Conventional Methods ◽  
Insertion Mutations

AbstractSite-directed mutagenesis for large plasmids is a difficult task that cannot easily be solved by the conventional methods used in many laboratories. In this study, we developed an effective method for Site-directed Mutagenesis for Large Plasmids (SMLP) based on a PCR technique. The SMLP method combines several effective approaches, including a high-efficiency DNA polymerase for the large DNA amplification, two independent PCR reactions and a fast recombinational ligation. Using this method, we have achieved a variety of mutants for the filamin A gene (7.9 kb) cloned in the pcDNA (5.4 kb) or the pLV-U6-CMV-EGFP (9.4 kb) plasmids, indicating that this method can be applied to site-directed mutagenesis for the plasmids up to 17.3 kb. We show that the SMLP method has a greater advantage than the conventional methods tested in this study, and this method can be applied to substitution, deletion, and insertion mutations for both large and small plasmids as well as the assembly of three fragments from PCR reactions. Altogether, the SMLP method is simple, effective, and beneficial to the laboratories that require completing the mutagenesis of large plasmids.

scholarly journals Parameters influencing high-efficiency transfection of bacterial artificial chromosomes into cultured mammalian cells

BioTechniques ◽  
2003 ◽  
Vol 35 (4) ◽  
pp. 796-807 ◽  
Author(s):  
William J. Montigny ◽  
Stephanie F. Phelps ◽  
Sharon Illenye ◽  
Nicholas H. Heintz
Keyword(s):  
Mammalian Cells ◽  
High Efficiency ◽  
Bacterial Artificial Chromosomes ◽  
Artificial Chromosomes

A high efficiency method for site-directed mutagenesis with any plasmid

Gene ◽  
1989 ◽  
Vol 84 (1) ◽  
pp. 153-157 ◽  
Author(s):  
Bernd Hofer ◽  
Birgit Kiihlein
Keyword(s):  
High Efficiency ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

scholarly journals Rapid and high efficiency site-directed mutagenesis by improvement of the homologous recombination technique

1995 ◽  
Vol 23 (9) ◽  
pp. 1642-1643 ◽  
Author(s):  
Annick Martin ◽  
Esther Toselli ◽  
Marie-Françoise Rosier ◽  
Charles Auffray ◽  
Marie-Dominique Devignes
Keyword(s):  
Homologous Recombination ◽  
High Efficiency ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis
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