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

doi:10.1016/j.xpro.2021.100907

MegaGate: A toxin-less gateway molecular cloning tool

STAR Protoc. 2021 Oct 22;2(4):100907. doi: 10.1016/j.xpro.2021.100907. eCollection 2021 Dec 17.

Authors

Christian Kramme^{1

2}, Alexandru M Plesa^{1

2}, Helen H Wang^{1

2}, Bennett Wolf^{1

2}, Merrick Pierson Smela^{1

2}, Xiaoge Guo^{1

2}, Richie E Kohman^{1

2}, Pranam Chatterjee^{1

2}, George M Church^{1

2}

Affiliations

¹ Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
² Wyss Institute for Biologically Inspired Engineering, Harvard University, 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).

Keywords: High-Throughput Screening; Molecular Biology; Stem Cells.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cloning, Molecular / methods*
DNA, Complementary / genetics
Escherichia coli / genetics
Gene Library
Plasmids / genetics
Polymerase Chain Reaction / methods*
Recombinant Fusion Proteins* / genetics
Recombinant Fusion Proteins* / metabolism

Substances

DNA, Complementary
Recombinant Fusion Proteins