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

Temperatures follow the selected enzyme's recommended settings; times and cycle counts are editable. The final hold is fixed at 4°C indefinitely. Estimated run time assumes a 3°C/sec block ramp rate and a 5 min initial heat-up.

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. Add parts from CSV to bulk-import a spreadsheet (columns: name, description, length in bp, and an optional 4th column — "acceptor" or "insert", defaults to insert). Add from GenBank reads one or more .gb/.gbk files' LOCUS/DEFINITION/ORIGIN (e.g. downloaded from Addgene or a sequencing provider) — select a single file with one record to review it in the form below first, or select multiple files/a multi-record file to import them all straight away. Only the name, description and calculated length are kept, not the sequence itself.

These parts are cached in this browser only — they are not stored on a server or synced anywhere. Clearing your browser data will delete them. Use Download backup regularly and keep the file somewhere safe; you are responsible for your own backups.

Name	Category	Length	Details