BioDeviceHub

Golden Gate Assembly Calculator

Plan a Golden Gate assembly: the picomoles, nanograms and volume of the destination 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 destination vector
rmolarr_{\text{molar}}
insert-to-vector molar ratio, applied to every insert

How it works

Golden Gate assembly uses a Type IIS restriction enzyme such as BsaI, BsmBI or BbsI, which cuts outside its recognition sequence and so can leave any chosen four-base overhang. Fragments with matching overhangs are ligated in the same tube, and because the joined product no longer has the recognition site, it is not cut again.

The planning is again in moles. A small destination vector amount, often a few tens of nanograms, with a molar excess of each insert, is typical. The page calculates each fragment's mass and volume, and checks they fit in the space the kit leaves for DNA.

Worked example

A 4,000 bp destination vector at 50 ng/µL and two inserts of 1,200 and 800 bp, 0.023 pmol of vector and a 2 : 1 insert ratio.

  1. Vector: 0.023 pmol = 59.8 ng = 1.20 µL.
  2. Insert 1: 0.046 pmol = 35.9 ng = 1.20 µL.
  3. Insert 2: 0.046 pmol = 23.9 ng = 0.96 µL.

3.35 µL of DNA in total, 0.115 pmol, well within the space available.

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

Assumptions

  • Every fragment carries the recognition sites and overhangs the enzyme needs, and none has an internal site for it.
  • Concentrations are accurate.
  • The defaults are starting values; follow the kit protocol for volumes and cycling.

Common mistakes

  • Forgetting that an insert with an internal recognition site is cut to pieces. Check each fragment for the enzyme's site.
  • Using overhangs that are palindromic or that appear twice, so the wrong fragments join.
  • Skipping the final enzyme-inactivation or heat step the protocol specifies.

Related tools

Restriction Site FinderSites, cut positions and overhangs for named restriction enzymesOpen calculatorGibson Assembly CalculatorFragment amounts and volumes for an overlap assemblyOpen 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 calculator

Related equipment

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