About this Video

• Video Title: A Detailed Look at Golden Gate Assembly
• Channel: SnapGene
• Speakers: None specified
• Duration: 00:05:19

Introduction

This video provides a detailed explanation of Golden Gate Assembly, a versatile cloning technique used to seamlessly assemble multiple DNA fragments in a single reaction. It focuses on the features of Type IIS restriction enzymes that make Golden Gate Assembly possible and efficient.

Key Takeaways

• Golden Gate Assembly utilizes Type IIS restriction enzymes: These enzymes possess non-palindromic recognition sites, cleave outside these sites creating variable overhangs, and leave behind no recognition sites after cleavage.
• Type IIS enzymes generate unique overhangs (fusion sites): These overhangs are crucial for the assembly process. Commonly used enzymes like BsaI create four-nucleotide overhangs leading to many possible combinations.
• Orientation of restriction sites is critical: In the destination vector, Type IIS sites must be outward-facing to allow for excision of the target region and prevent self-ligation. In the insert fragments, sites must be inward-facing.
• Golden Gate Assembly is a two-phase process: It involves alternating cycles of digestion, annealing, and ligation within a single reaction. The thermocycler controls the temperature shifts optimizing each phase.
• PCR can be used to adapt fragments for Golden Gate Assembly: PCR primers can be designed to incorporate the necessary Type IIS recognition sites and overhang sequences.

The video describes Golden Gate Assembly as a two-phase process occurring within a single reaction, but doesn't explicitly list steps in a numbered format. However, based on the transcript, here's a summary of the process broken down into steps:

1. Preparation: Design and prepare the destination vector (containing outward-facing Type IIS restriction sites flanking the insertion region) and the DNA fragments of interest (containing inward-facing Type IIS restriction sites). PCR may be used to add the necessary restriction sites to your fragments. Ensure any disruptive Type IIS sites within your insert are removed.

2. Mixing: Combine the prepared destination vector and DNA fragments in a single reaction tube with DNA ligase and the appropriate Type IIS restriction enzyme.

3. Thermocycling: Subject the reaction mixture to a thermocycling protocol that alternates between temperatures optimal for enzyme digestion, annealing (of complementary overhangs), and ligation. This cycling repeats to increase the likelihood of the complete desired construct being generated.

4. Digestion (Final): A final digestion step with the Type IIS enzyme eliminates any remaining unmodified destination vector that didn't participate in the assembly.

5. Enzyme Inactivation: Heat inactivation of the enzyme at 80°C.

6. Transformation: The assembled plasmid is then ready for transformation into an appropriate host.