MegaGate: A toxin-less gateway molecular cloning tool

Gateway cloning employs the use of the ccdb toxin and has low colony numbers, making it difficult to apply at scale to clone libraries of cDNA vectors. In this protocol, we describe MegaGate, a toxin-less Gateway technology capable of robust cDNA library cloning that is efficient, cheap, and scalabl...

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Detalles Bibliográficos
Autores principales: Kramme, Christian, Plesa, Alexandru M., Wang, Helen H., Wolf, Bennett, Smela, Merrick Pierson, Guo, Xiaoge, Kohman, Richie E., Chatterjee, Pranam, Church, George M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2021
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8551244/
https://www.ncbi.nlm.nih.gov/pubmed/34746865
http://dx.doi.org/10.1016/j.xpro.2021.100907
Descripción
Sumario:Gateway cloning employs the use of the ccdb toxin and has low colony numbers, making it difficult to apply at scale to clone libraries of cDNA vectors. In this protocol, we describe MegaGate, a toxin-less Gateway technology capable of robust cDNA library cloning that is efficient, cheap, and scalable. MegaGate eliminates the ccdb toxin used in Gateway recombinase cloning and instead utilizes meganuclease-mediated digestion to eliminate background vectors during cloning and is 99.8% efficient with high colony numbers. For complete details on the use and execution of this protocol, please refer to Kramme et al. (2021).

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