What this is for: Insert mass and volume for ligations at a chosen vector:insert molar ratio, plus a pmol-based Gibson/HiFi assembly sheet.
The formula: insert (ng) = vector (ng) × (insert kb ÷ vector kb) × molar ratio; Gibson: ng = pmol × kb × 650
How to use:
- For a ligation, enter the vector length (kb), vector amount to use (ng) and vector concentration (ng/µL), plus the insert length (kb) and insert concentration (ng/µL).
- Read the table by molar ratio: insert needed (ng), insert stock (µL), vector stock (µL) and both amounts in pmol — the 3:1 row is the usual starting point.
- For Gibson / HiFi, scroll to the second section and enter its vector, insert, target vector amount (pmol), molar ratio, 2X master mix (µL) and total reaction volume (µL).
- Read the vector and insert needed in ng and µL, and the water to total (µL), in that section.
Ligation — Insert:Vector Molar Ratios
ng insert = ng vector × (kb insert / kb vector) × molar ratio. pmol = ng × 1000 / (bp × 650). The 3:1 row is the usual starting point.
Vector volume to pipette (µL)
Vector amount (pmol)
Insert needed at 3:1 (ng)
Insert stock at 3:1 (µL)
Gibson / HiFi Assembly — 2-Fragment Molar Calculator
A common target is 0.05 pmol of vector; inserts are usually added at a 2:1 insert:vector molar ratio for one insert. ng = pmol × bp × 650 / 1000.
Vector needed (ng)
Vector stock (µL)
Insert needed (pmol)
Insert needed (ng)
Insert stock (µL)
Water to total (µL)
Prefer to work offline or keep a copy? The download link is right below.
Download the Ligation & Gibson/HiFi Calculator workbook (.xlsx)
Download the Ligation & Gibson/HiFi Calculator workbook (.xlsx)
Notes & limits (from the workbook): ng insert = ng vector × (kb insert / kb vector) × molar ratio. pmol = ng × 1000 / (bp × 650). Rule of thumb: 50–100 ng vector per ligation; 3:1 for cohesive ends, up to 10:1 for blunt ends. Gibson/HiFi: a common target is 0.05 pmol of vector; inserts are usually added at a 2:1 insert:vector molar ratio for one insert. ng = pmol × bp × 650 / 1000.