Golden Gate Assembly Calculator

Assemblies

Master mix

Use a shared master mix across assemblies

Global conditions

Acceptor target FMOL Insert target (each) FMOL Reaction volume µL

Volume calculation

Sequence composition (exact) Length only (bp average — less accurate)

Master mix reagents (used per reaction, individually or batched)

Recommended cycling

Ramp rate °C/sec Initial heat-up min

Temperatures follow the selected enzyme's recommended settings; times and cycle counts are editable.

Why 20–30 cycles is often enough

Efficiency blue colonies / 100 µl outgrowth

Accuracy % colonies with correct insert

Average Duplicate range ~30 cycles

Efficiency keeps climbing through 60 cycles, but accuracy plateaus around 90–93% by roughly 30 cycles — cycles beyond that mainly buy more colonies, not more correct ones. Values are approximate, read from an internal two-duplicate pilot; illustrative only.

Show data table

Cycles	Efficiency avg (range)	Accuracy avg (range)
0	0	0%
15	16 (11–21)	80% (65–95%)
30	53 (49–58)	91% (88–94%)
45	96 (93–99)	93% (91–95%)
60	146 (136–155)	93.5% (92–95%)

My saved parts

Remember a fragment by name and length when you don't have (or don't need) the full sequence — handy for backbones and parts the lab reuses often. Saved parts appear here and in the search below, and are stored only in this browser — use Download/Upload to back them up or move them to another browser.

Name	Category	Length	Details