MegaGate: A toxin-less gateway molecular cloning tool

Christian Kramme; Alexandru M. Plesa; Helen H. Wang; Bennett Wolf; Merrick Pierson Smela; Xiaoge Guo; Richie E. Kohman; Pranam Chatterjee; George M. Church

STAR Protocols (Dec 2021)

MegaGate: A toxin-less gateway molecular cloning tool

Christian Kramme,
Alexandru M. Plesa,
Helen H. Wang,
Bennett Wolf,
Merrick Pierson Smela,
Xiaoge Guo,
Richie E. Kohman,
Pranam Chatterjee,
George M. Church

Affiliations

Christian Kramme: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA
Alexandru M. Plesa: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA
Helen H. Wang: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA
Bennett Wolf: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA
Merrick Pierson Smela: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA
Xiaoge Guo: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA
Richie E. Kohman: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA
Pranam Chatterjee: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA; Corresponding author
George M. Church: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA

Journal volume & issue: Vol. 2, no. 4
p. 100907

Abstract

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Summary: 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).

Published in STAR Protocols

ISSN: 2666-1667 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Science: Science (General)
Website: https://star-protocols.cell.com/

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