NEB Gibson Assembly Calculator

Example Data Table

Formula Used

Picomoles from DNA mass:

pmol = DNA mass in ng ÷ (0.66 × fragment length in bp)

DNA mass from picomoles:

ng = target pmol × 0.66 × fragment length in bp

Fragment volume:

volume in µL = required ng ÷ concentration in ng/µL

Insert target:

insert pmol = vector pmol × desired insert molar ratio

Water volume:

water = final reaction volume − assembly mix volume − total DNA volume

Simple overlap temperature estimate:

estimated Tm = 2 × AT bases + 4 × GC bases

How to Use This Calculator

Enter the total reaction volume and assembly mix volume first. Add vector length, concentration, and target vector amount. Select the number of inserts used in the reaction. Then enter each active insert length, concentration, desired molar ratio, overlap length, and overlap GC value.

Press the submit button to view the result below the header and above the form. Review vector pmol, insert mass, insert volume, water volume, and warning messages. Use CSV export for spreadsheet records. Use PDF export for a printable setup sheet.

If a calculated volume is too small to pipette accurately, dilute the stock DNA or increase the reaction scale. If water becomes negative, lower DNA inputs, reduce mix volume only when allowed, or increase the total reaction volume.

Advanced NEB Gibson Assembly Planning

Gibson assembly works best when every fragment enters the reaction in a controlled molar amount. This calculator supports that planning step. It helps you convert DNA mass into picomoles, estimate insert volumes, review overlap lengths, and compare final reaction totals before mixing reagents. The goal is simple. You can reduce guesswork and prepare a cleaner assembly setup.

Why Molar Balance Matters

Fragments assemble by matching overlapping ends. A long backbone and a short insert may require very different nanogram amounts to supply the same number of molecules. Using mass alone can create excess insert or too little vector. The calculator corrects that problem by using length, concentration, and target molar ratios. It also reports total DNA mass, final liquid volume, and remaining master mix space.

Useful Inputs

Enter the vector length, insert length, DNA concentrations, target vector amount, desired insert ratio, overlap length, reaction volume, and assembly mix volume. Optional controls let you include more inserts, dilution water, and notes for tracking. The result area appears immediately after submission. It shows the main setup values first, so the page remains practical during bench preparation.

Practical Setup Guidance

For many Gibson reactions, overlaps between 15 and 40 bases are commonly useful. Very short overlaps may reduce specificity. Very long overlaps can increase primer cost. Fragment purity also matters. Salt, ethanol, primer dimers, and nonspecific bands can lower assembly performance. Use clean DNA and verify fragment sizes when possible.

Accuracy Notes

The tool uses standard double stranded DNA mass conversion. It assumes average base pair mass and linear fragments. It does not replace experimental judgment. Pipetting limits can make tiny volumes difficult. When calculated volumes fall below comfortable handling ranges, dilute stock DNA or increase the reaction scale. Record each adjustment, because repeatable notes make troubleshooting much easier later for future cloning sessions safely.

Export and Review

The CSV download is useful for spreadsheets, lab notebooks, or batch planning. The PDF download creates a printable summary for records. The example table gives realistic sample inputs for a backbone and insert. You can edit those values to match your own design. Always compare the calculated plan with your reagent instructions and internal lab procedures before starting the reaction.