Learn that Gibson assembly is a method developed by Dr. Daniel Gibson for assembling multiple DNA fragments in a single tube isothermal reaction using Gibson assembly mastermix.
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Prepare DNA fragments with overlaps
Prepare multiple DNA fragments with complementary overlapping ends using PCR or other methods, then add them to the Gibson assembly mastermix.
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Incubate reaction at fifty degrees
Incubate the DNA fragments and Gibson assembly mastermix in a single tube at 50°C for 1 hour to initiate the enzymatic assembly process.
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Create single-stranded overhangs enzymatically
The exonuclease activity in the mastermix creates single-stranded 3' overhangs that are complementary, allowing the DNA fragments to anneal together.
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Fill gaps and seal DNA strands
DNA polymerase extends the 3' ends to fill gaps, and DNA ligase seals the remaining nicks to create a fully sealed double-stranded DNA molecule.
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Use assembled DNA for applications
The final assembled double-stranded DNA product can be used as a template for PCR, RCA, transformation, or other molecular biology applications.
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Apply Gibson assembly for cloning
Use the Gibson assembly cloning kit containing mastermix and competent cells to efficiently assemble and clone multiple DNA fragments into a vector within 2 hours.
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