Gibson Assembly
Course Content
Gibson Assembly with SnapGene
Gibson Assembly with Multiple Inserts
Common Questions About Using SnapGene
What does SnapGene's Gibson Assembly simulation produce?
The simulation designs PCR primers with overlapping ends, assembles the linearised vector and insert(s), and generates a product file. The History view records the PCR and assembly steps. History colors show exactly where each fragment was incorporated: red for a single insert, alternating red and blue for multiple inserts.
How does SnapGene design primers for Gibson Assembly?
In the Product tab, clicking Choose Overlapping PCR Primers lets you set the target Tm for the PCR primers, as well as the desired overlap length and Tm for the Gibson Assembly reaction. SnapGene then designs primers with appropriate overlapping tails for each fragment.
Can SnapGene regenerate restriction sites after Gibson Assembly?
Yes. When setting up the simulation, you can choose to regenerate one or both halves of the restriction site used to linearise the vector by including it in the PCR primers. You can also choose to remove the partial restriction site entirely if it is not needed in the product.
Can SnapGene simulate Gibson Assembly to circularise a linear fragment?
Yes. The Gibson Assembly ‘Assemble Multiple Fragments’ option can be used to circularize a single fragment. You specify the fragment, set overlap parameters, and check the option to Generate a circular product. SnapGene designs appropriate primers and simulates the assembly.
Can SnapGene simulate multi-insert Gibson Assembly?
Yes. The second video in this series covers multi-insert assembly. Each insert is defined separately with its own overlap parameters. In the Product tab, Sequence view allows you to scroll through and check all fusion boundaries, including reading frame continuity at each junction.
What is the difference between Gibson Assembly and NEBuilder HiFi Assembly simulations in SnapGene?
Both methods use overlapping ends and follow similar workflows in SnapGene. The NEBuilder HiFi Assembly tool includes a separate Tm setting for the HiFi assembly reaction itself, in addition to the PCR primer Tm. NEBuilder HiFi Assembly also supports assembly from single-stranded oligonucleotide inserts. The two are accessed via separate tools under Actions.
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