Simulate NEBuilder HiFi Assembly

HiFi Assembly offers a fast and accurate method for joining DNA fragments using short, high-fidelity overlaps. CodonCode Aligner supports this approach with intuitive tools to design overlaps, assemble fragments in silico, and review completed constructs before moving to the lab. While HiFi Assembly works similarly to Gibson Assembly, it uses optimized reagents and shorter overlaps, providing a more streamlined option for certain cloning projects. This page gives a brief overview of how HiFi Assembly works in Aligner, along with screenshots and tips to help ensure reliable cloning results.

How HiFi Assembly Works in CodonCode Aligner

Find out how to use the virtual HiFi cloning tool in CodonCode Aligner.

Start HiFi Assembly

Start the cloning wizard by using the "HiFi DNA Assembly..." feature in the "Tools" menu. It will guide you through the single steps.

Select a Vector

Select the vector, choose how to linearize it, and which part of the sequence to use. Selections can also easily be made via the sequence map.

Select One or More Fragments

HiFi DNA Assembly can be done with one or multiple fragments. Select the fragments in the cloning wizard and verify the lengths.

Pick Primers

Automatically design the best cloning primers for your HiFi Assembly. Preview the primers along with their Tm, length, overlap and annealing sequences.

Create Cloning Product

Simulate the HiFi Assembly. Double check your product visually, looking at features, insert orientations, and verify the sources.

Document & Verify

Create all sequences in silico, check the cloning history for an overview and summary of all steps, and use clone verification to verify your HiFi Assembly results.

Best Practices and Tips for HiFi DNA Assembly in CodonCode Aligner

HiFi DNA Assembly is generally robust and efficient, but careful planning and preparation can greatly improve cloning success. Below are practical tips to help you get the best results in the lab and when planning assemblies in CodonCode Aligner.

• Favor Precise, High-Quality Overlaps (15–20 bp)
  HiFi mixes perform optimally when overlaps are well-defined and error-free. Short but accurate overlaps improve both fidelity and assembly efficiency.
• Prefer HiFi Assembly for GC-Rich or Difficult Regions
  HiFi polymerases handle GC-rich fragments and secondary structures more robustly than standard Gibson formulations. If you see mis-assemblies with Gibson, HiFi can often resolve them.
• Use HiFi for Multi-Fragment Projects
  HiFi mixes typically show good efficiency as fragment count increases (e.g. 3–6 fragments). Use HiFi DNA Assembly when accuracy and complexity matter.
• Allow Slightly Longer Extension Times for Large Inserts
  HiFi polymerases extend more accurately but sometimes slightly slower. A small buffer in incubation time can improve yields for large (> 2–3 kb) fragments.
• Maintain Balanced Fragment Concentrations
  Equimolar amounts are a good default. Avoid large molar excess of vector or the smallest fragments.

Explore Other Cloning Methods and Features in CodonCode Aligner

CodonCode Aligner supports a variety of molecular cloning techniques beyond HiFi DNA Assembly. Browse the pages below to learn how Aligner can help you design, simulate, and verify different cloning projects:

• Clone Verification
  Automatic clone verification: Quickly group your reads by clones, match them to a reference, and verify if the sequences match your expected clone.
• Gibson Assembly
  Seamless DNA assembly without the need for restriction enzymes — ideal for multi-fragment cloning and scarless constructs.
• Restriction Cloning
  Simulate restriction enzyme digests, ligation, and insert orientation with support for hundreds of enzymes.
• TA Cloning
  Learn how to simulate TA cloning and verify insert orientation before going to the bench.
• Blunt-End PCR Cloning
  Plan and test blunt-end ligation of PCR products and vectors, with annotation and map preview features.
• Directional TOPO Cloning
  Learn how to simulate directional TOPO cloning and verify insert orientation before going to the bench.
• Cloning History
  See how the cloning history automatically documents how each construct was createded.
• Molecular Cloning
  Overview of virtual molecular cloning methods in CodonCode Aligner.
• Primer Design
  Design primers using Primer3. Adjust parameters like primer characteristics and reaction conditions to create PCR, cloning, and sequencing primers that specifically match your workflow.
• Virtual Gels and RFLP Analysis
  Use virtual gels to verify and plan any gels you use in the lab. You can look at restriction maps and virtual gels for single sequences, or even compare multiple sequences (for example your cloning product in both directions) using the RFLP analysis tool in Aligner.