MegaGate: A toxin-less gateway molecular cloning tool
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Updated: Oct 14, 2025
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Published on: October 31, 2019
Christian Kramme1,2, Alexandru M Plesa1,2, Helen H Wang1,2
1Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
Abstract:
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).
