Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

Minakshi Mukherjee; Emily Caroll; Zhen Q. Wang

doi:10.3791/61993

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

JoVE Journal
Bioengineering

A subscription to JoVE is required to view this content. Sign in or start your free trial.

JoVE Journal Bioengineering

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

Full Text

15,005 Views

08:31 min

February 5, 2021

DOI: 10.3791/61993-v

Minakshi Mukherjee*¹, Emily Caroll*¹, Zhen Q. Wang¹

¹Department of Biological Sciences, University at Buffalo,State University of New York

The goal of this protocol is to provide a detailed, step-by-step guide for assembling multi-gene constructs using the modular cloning system based on Golden Gate cloning. It also gives recommendations on critical steps to ensure optimal assembly based on our experiences.

This is a detailed protocol for assembling multi-gene plasmids using the Yeast MoClo kit. This method enables the convenient cloning of a variety of multi-gene classmates. It is ideal for generating a big library of related parts.

Begin by setting up the Golden Gate reaction mix. Add 20 femtomoles of each PCR product and the entry vector, one microliter of 10X T4 Ligase buffer, 0.5 microliters of Esp3I, and 0.5 microliters of T4 ligase. Add double-distilled water to bring the total volume to 10 microliters.

View the full transcript and gain access to thousands of scientific videos