Gibson assembly calculator
Insert and vector amounts at your molar ratio, plus an overlap Tm check.
Vector
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Insert
Insert and vector amounts at your molar ratio, plus an overlap Tm check.
Gibson assembly joins two or more linear DNA fragments that share 15 to 40 bp of identical sequence at their ends. In one isothermal reaction at 50 °C, a 5′ exonuclease exposes the overlaps as single strands, the complementary ends anneal, a polymerase fills the gaps and a ligase seals the nicks. The result is a seamless construct with no restriction sites or scars. NEBuilder HiFi DNA Assembly uses the same principle and the same amounts.
Fragments are combined by molar ratio, not by mass, because a short insert has far more molecules per nanogram than a long vector. For a given vector mass the insert mass is:
Worked example. 50 ng of a 5,000 bp vector with a 1,500 bp insert at 2:1: 50 × (1,500 ÷ 5,000) × 2 = 30 ng of insert. At 25 ng/µL that is 1.2 µL. The calculator repeats this for every insert, so a 4-fragment assembly is just three insert rows.
Suppliers state their recommended amounts in picomoles. Double-stranded DNA weighs about 650 g/mol per base pair, so:
50 ng of a 5,000 bp vector is 0.015 pmol. NEB recommends 0.02 to 0.5 pmol per fragment for 2 to 3 fragments, and 0.2 to 1.0 pmol each for 4 to 6 fragments, with the total DNA kept under about 200 ng in a 20 µL reaction. The table above shows pmol for every fragment so you can check against those ranges.
For conventional sticky-end cloning the same molar logic applies; see theligation calculator and thevector to insert ratio tool.
Each junction needs 15 to 40 bp of identical sequence on both fragments. 20 to 25 bp is the sweet spot. Aim for 40 to 60 % GC and a Tm of at least 48 °C so the annealed ends stay paired at 50 °C. Avoid runs of five or more of the same base, and keep the overlap free of repeats that could pair with the wrong fragment. The overlap check uses the Wallace rule for overlaps under 14 bp and the GC-content formula Tm = 64.9 + 41 × (GC − 16.4) ÷ N for longer ones.
Scale the insert mass by size and ratio: insert ng = vector ng × (insert bp ÷ vector bp) × molar ratio. For 50 ng of a 5,000 bp vector and a 1,500 bp insert at 2:1 that is 30 ng of insert.
NEB recommends 2:1 to 3:1 insert to vector for 2 to 3 fragments, and equimolar (1:1) for 4 to 6 fragments. Use 0.02 to 0.5 pmol of each fragment for 2 to 3 fragments and 0.2 to 1 pmol each for 4 to 6.
pmol = ng × 1000 ÷ (650 × length in bp). A base pair of double-stranded DNA weighs about 650 g/mol, so 1 pmol of a 1,000 bp fragment is 650 ng.
15 to 40 bp, ideally 20 to 25 bp with 40 to 60 % GC and a Tm of at least 48 °C, since the reaction runs at 50 °C. Avoid runs of five or more identical bases in the overlap.
Yes. NEBuilder HiFi uses the same molar-ratio and overlap rules as classic Gibson assembly, so the same amounts apply.