Gibson Assembly Calculator

🧩 Fragments

input calculated

	Label	Length bp	Conc. ng/µL	Fold excess optional	Volume µL	Mass ng	Amount pmol	Fold used
	Water (H₂O)				—
	Total				—		—

⚗️ Reaction parameters

v_max — volume for fragments (µL) 5 µL leaves room for 1.33× master mix. Default insert fold excess Blank = NEB defaults (5× ≤ 200 bp, else 2.5×). Set a custom molar ratio for all inserts. Master mix — reactions (N) Scale volumes for a master mix of N reactions in the export & protocol.

m_max 100 ng Max vector mass (fixed).

p_max 0.5 pmol 0.5 pmol for 2–3 frags, 1 pmol for 4+.

m₁ — vector mass — min of the three limits (see below).

🧪 Master mix (1× reactions)

Per-fragment and water volumes scaled for 1 reactions. Prepare a single mix, then aliquot.

Component	Volume ×N µL
Total	—

🧪 How to use

Enter the length and concentration for each fragment in the green cells.
Labels are optional and may be overwritten.
By default, NEB-recommended molar fold excesses are used. If there is no vector fragment, use the largest fragment as the “vector” (row 1): its fold excess is 1, and you can manually set the fold excesses of the other fragments.
Read off the volume to pipette for each fragment and for water (blue cells).

🤓 How it works

The volume of the n-th fragment is

v_n = ( f_n · l_n / c_n ) · ( m₁ / l₁ ), where m₁ = min( l₁·v_max / Σ (f_nl_n/c_n), φ·l₁·p_max / Σ f_n, m_max )

n	number of fragments
v_n	volume of n-th fragment (µL)
f_n	molar fold excess of n-th fragment
l_n	length of n-th fragment (bp)
l₁	length of the vector (bp)
m_n	mass of n-th fragment (ng)
m₁	mass of the vector (ng)
c_n	concentration of n-th fragment (ng/µL)
v_max	volume available for fragments (5 µL for 1.33× master mix)
m_max	optimally 100 ng
p_max	optimally 0.5 pmol (2–3 frags) or 1 pmol (4+ frags)
φ	0.66 ng/pmol/bp (conversion factor)