Calculator inputs
Example data table
These example setups show how fragment size and stock concentration influence the required insert mass and pipetting volume.
| Example | Vector (bp) | Insert (bp) | Target ratio | Vector DNA (ng) | Required insert (ng) | Vector conc. (ng/µL) | Insert conc. (ng/µL) |
|---|---|---|---|---|---|---|---|
| Standard subcloning | 3000 | 1000 | 3:1 | 50 | 50.00 | 25 | 20 |
| Small insert cloning | 5000 | 250 | 5:1 | 40 | 10.00 | 20 | 10 |
| Large fragment assembly | 4500 | 2200 | 2:1 | 60 | 58.67 | 30 | 25 |
| Low-concentration insert | 2800 | 900 | 4:1 | 30 | 38.57 | 15 | 6 |
Formula used
Insert ng = Vector ng × (Insert bp ÷ Vector bp) × (Insert ratio ÷ Vector ratio)
pmol = (ng × 1000) ÷ (fragment length × molecular weight per base unit)
Volume (µL) = DNA mass (ng) ÷ DNA concentration (ng/µL)
Water = Final reaction volume − all other component volumes
For double-stranded DNA, the calculator uses an average molecular weight of 650 g/mol per base pair. For single-stranded DNA, it uses 330 g/mol per base. These are standard approximations used for molar conversion.
This calculator estimates stoichiometric balance only. End compatibility, phosphorylation state, ligase choice, incubation conditions, and vector background still influence real cloning outcomes.
How to use this calculator
- Enter the vector and insert fragment sizes in base pairs.
- Set the vector DNA mass you want to use in one ligation.
- Provide stock concentrations for vector and insert DNA.
- Choose the desired insert:vector molar ratio, such as 3:1.
- Enter reaction volume, buffer, ligase, and any extra reagent volume.
- Set replicate count and pipetting overage if preparing a master mix.
- Submit the form to view required insert mass, pmoles, component volumes, and master mix totals.
- Use the CSV or PDF buttons to export a record for lab planning.
FAQs
1) What does a ligation ratio mean?
A ligation ratio expresses the molar relationship between insert molecules and vector molecules. A 3:1 ratio means three insert molecules are targeted for every one vector molecule in the reaction.
2) Why is molar ratio better than mass ratio?
Fragments with different lengths have different molecular counts at the same mass. Molar ratio corrects for length, so the reaction is balanced by molecule number instead of raw nanograms.
3) What ratio should I start with?
A 3:1 insert:vector ratio is a common starting point for routine cloning. Larger inserts, blunt ends, or low-efficiency systems may need optimization across several ratios.
4) Does this calculator account for sticky or blunt ends?
No. It estimates stoichiometry only. Sticky ends, blunt ends, compatible overhangs, and phosphorylation state can change ligation efficiency even when the molar ratio is correct.
5) Why is my water volume negative?
A negative water value means the combined DNA and reagent volumes exceed the final reaction volume. Increase the total volume or use more concentrated DNA stocks.
6) Can I use this for master mix planning?
Yes. Enter the number of replicate reactions and an overage percentage. The calculator scales all reagent amounts so you can prepare one larger mix with pipetting allowance.
7) Why does insert concentration matter?
Concentration affects the pipetting volume needed to deliver the required insert mass. Low-concentration inserts can consume too much reaction volume and reduce setup flexibility.
8) Is the molecular weight conversion exact?
No. The calculator uses standard average molecular weight approximations. They are suitable for planning ligations, but exact molecule mass can vary slightly with sequence composition and modifications.