BioDeviceHub

Gibson Assembly Calculator

Plan a Gibson or HiFi-style overlap assembly: the picomoles, nanograms and volume of the vector and each insert for a chosen vector amount and insert ratio.

Formula

pmol=m (ng)×1000L (bp)×650\mathrm{pmol} = \dfrac{m\,(\mathrm{ng})\times 1000}{L\,(\mathrm{bp})\times 650}
pmolinsert=pmolvector×rmolar\mathrm{pmol}_{\text{insert}} = \mathrm{pmol}_{\text{vector}} \times r_{\text{molar}}
V=mCV = \dfrac{m}{C}
pmolvector\mathrm{pmol}_{\text{vector}}
pmol of the linearised vector
rmolarr_{\text{molar}}
insert-to-vector molar ratio, applied to every insert

How it works

Gibson assembly joins fragments that share overlapping ends, in a single isothermal reaction. As in ligation, the planning is done in moles, so that each fragment is present in the ratio intended. Each fragment's mass follows from its length and the picomoles wanted, and its volume from its concentration.

Kit guidance commonly gives a total DNA amount and a vector-to-insert ratio for two or three fragments, and a different ratio for very short fragments. The defaults here are commonly used starting values, editable, and the page checks that the DNA fits in the volume the reaction has room for.

Worked example

A 4,000 bp vector at 50 ng/µL with inserts of 1,200 bp (30 ng/µL) and 800 bp (25 ng/µL), 0.05 pmol of vector and a 2 : 1 insert ratio.

  1. Vector: 0.05 pmol = 130.0 ng = 2.60 µL.
  2. Insert 1: 0.10 pmol = 78.0 ng = 2.60 µL.
  3. Insert 2: 0.10 pmol = 52.0 ng = 2.08 µL.

7.28 µL of DNA in total, 0.25 pmol, which fits in the 10 µL available.

These are the values the calculator opens with, so you can check its output against this example.

Assumptions

  • Double-stranded fragments of the lengths entered, with overlaps already designed into their ends.
  • Concentrations are accurate and the fragments are clean, since carried-over salt or polymerase inhibits the reaction.
  • The defaults are starting values; the kit's own protocol sets the total volume and number of fragments.

Common mistakes

  • Using masses instead of moles, which starves the vector of inserts or swamps it.
  • Leaving template plasmid in a PCR fragment, which produces background colonies. Digest it with DpnI or gel-purify.
  • Designing overlaps without checking for secondary structure that blocks annealing.

Related tools

Homology Arm CalculatorExtract left and right homology arms around an edit siteOpen calculatorLigation Calculator (Vector:Insert Ratio)Insert mass for a molar ratio, or the ratio from the masses you haveOpen calculatorPlasmid Size CalculatorSize of a recombinant plasmid after cloningOpen calculatorGolden Gate Assembly CalculatorFragment amounts and volumes for a Type IIS assemblyOpen calculator

Related equipment

Service documentation, failure modes and parts for the instruments this calculation is used with.