Team:Queens Canada/gibson

gBlock Gibson Assembly

Introduction

The Gibson Assembly Method described by Gibson is a rapid assembly method that provides directional cloning of multiple DNA fragments in a single reaction, without the need for specific restriction sequences. It relies on use of an enzyme mixture consisting of a mesophilic exonuclease, a thermophilic ligase, and a high-fidelity polymerase. For the assembly reaction, the gBlocks Gene Fragments and the vector insertion site are designed with overlapping sequences at the locations that are to be joined. At 50˚C, the exonuclease digests dsDNA from the 5’ ends, but is rapidly degraded leaving complementary, 3’ ssDNA ends. The resulting single-stranded, complementary ends are then availible to hybridize to each other, at which point the polymerase fills in missing nucleotides and the ligase covalently joins the fragments together.

Materials

• gBlocks Gene Fragments (with 20-80 bases of sequence overlaps)
• Linearized plasmid
• Gibson Assembly Master Mix (neb)
• Cell transformation reagents

Procedure

• IDT

1. Two or more gBlocks Gene Fragments are designed with 20-80 base overlapes with the adjacent gBlocks fragment sequences and the linearized plasmid. The plasmid can be linearized by restriction digest or PCR.

2. Linearized plasmid and fragments are combined in a tube with Gibson Assembly Master Mix.

3. Incubate at 50C for 1 hr.

4. The resulting completed plasmid is ready for transformation into the bacteria, and sequencing. Note: IDT scientists recommend sequencing at least 2X the number of gBlocks Gene Fragments assembled. For example, if you assembled 2 fragments, you would sequence four clones.