Calculate optimal fragment volumes for Gibson Assembly and HiFi DNA Assembly reactions. Equimolar or max-input modes, per-fragment volume tables, incubation guidance, and printable protocol. Supports 2–15 fragment assemblies.
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Minimum 120 bp per fragment. Exactly one fragment must be designated as the vector.
When to use
Do not use for
NEB recommends 15 minutes incubation for 2-fragment assemblies and 60 minutes for 3+ fragments. This calculator automatically adjusts the protocol based on your fragment count.
Accurate fragment quantification (e.g. by Nanodrop or Qubit) is critical. A 2-fold error in concentration means a 2-fold error in molar ratio, which can drastically reduce assembly efficiency.
For difficult assemblies, try max-input mode which uses a 2:1 insert:vector molar ratio. This can improve colony counts when insert concentration is limiting.
Gibson Assembly Master Mix is 2X, so fragments must fit in half the reaction volume (10 uL for a 20 uL reaction). If fragment volumes exceed this, concentrate your DNA or reduce fragment count.
If your vector was prepared by PCR from a dam+ template, add DpnI to digest the methylated template before assembly. Template carryover is the #1 cause of false-positive colonies.
Fragment MW = length (bp) × 650 Da/bp. Target pmol per fragment adjusted by assembly mode (equimolar or 2:1 insert:vector). Volume = (target_pmol × MW) / (concentration × ). Volumes rounded to 0.1 µL (lab-realistic pipetting precision). Protocol follows NEB Gibson Assembly and NEBuilder HiFi recommendations. Gibson Assembly is a method developed by Daniel G. Gibson. NEBuilder HiFi is a trademark of New England Biolabs, Inc. This tool is independent and not affiliated with NEB.
Last validated 2026-04-07. Calculations are designed for planning and documentation support; verify procurement decisions against manufacturer specifications or institutional SOPs.
ConductScience Gibson Assembly Reaction Calculator (v1.16.0). ConductScience, Inc. 2026. Available at: https://conductscience.com/tools/gibson-assembly-reaction-calculator
Gibson DG et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods. 2009;6:343–345. doi:10.1038/nmeth.1318
NEB. NEBuilder HiFi DNA Assembly Protocol (E2621). New England Biolabs. 2024. Available at: https://nebuildercalculator.neb.com/
Gibson Assembly (Gibson et al., 2009) joins multiple DNA fragments with overlapping ends in a single isothermal reaction at 50°C. The reaction contains three enzymes:
1. T5 exonuclease — chews back 5′ ends, exposing complementary 3′ overhangs 2. Phusion DNA polymerase — fills in gaps 3. Taq DNA ligase — seals nicks
The overlapping ends anneal, and the polymerase/ligase repair the junctions to produce a seamless, scar-free assembly.
The molecular weight of dsDNA is approximately 650 Da per base pair. To calculate pmol:
NEB recommends 0.02–0.5 pmol per fragment for Gibson Assembly. For a typical 2-fragment assembly: 0.1 pmol each. For 4+ fragments: 0.05 pmol each to keep total volume within the reaction.
Each fragment must share 15–40 bp of overlap with its neighbor. Key guidelines:
