NEBuilder HiFi Assembly
Course Content
NEBuilder HiFi Assembly in SnapGene
NEBuilder HiFi Assembly with Multiple Inserts
Common Questions About Using SnapGene
What does SnapGene's NEBuilder HiFi Assembly simulation cover?
The simulation designs overlapping PCR primers, sets overlap length and Tm for both the PCR step and the HiFi assembly reaction, and then generates a product file. History view records all simulated steps. The workflow mirrors the Gibson Assembly simulation but uses the NEBuilder HiFi enzyme system and includes a separate Tm parameter specific to the HiFi reaction.
Does SnapGene include a separate Tm setting for the NEBuilder HiFi assembly reaction?
Yes. The NEBuilder HiFi Assembly tool includes a dedicated Tm setting for the assembly reaction in addition to the PCR primer Tm. This distinguishes it from the Gibson Assembly tool, which lacks a separate assembly Tm field.
Can SnapGene simulate NEBuilder HiFi Assembly with a single-stranded insert?
Yes. A dedicated support article covers assembly with a single-stranded oligonucleotide insert, such as a CRISPR guide RNA, into a linearised vector. The oligo is loaded as the fragment source; if SnapGene identifies that it already has overlaps suitable for assembly, it proceeds directly to the Product tab without requiring primer design.
Can SnapGene retain or remove restriction sites when designing NEBuilder HiFi primers?
Yes. In the Product tab, you can check options to regenerate or remove partial restriction sites at the ends of the linearised vector before designing primers. This ensures restriction sites used for vector linearisation can be preserved or eliminated as needed in the final product.
Can SnapGene simulate NEBuilder HiFi Assembly to circularise a linear fragment?
Yes. The NEBuilder ‘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.
What is the difference between this simulation series and the Gibson Assembly simulation series?
Both series cover overlap-based seamless assembly and follow similar workflows in SnapGene. The NEBuilder HiFi series uses NEB's proprietary HiFi enzyme mix and includes a separate Tm parameter for assembly. It also supports single-stranded oligonucleotide inserts. The Gibson Assembly series follows the standard three-enzyme Gibson protocol. Both tools are available separately under Actions.
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