Calculate the insert DNA mass for a ligation from vector mass, fragment lengths and the insert:vector molar ratio. An optional reaction volume gives the final DNA concentrations.
Optionally enter vector and insert stock concentrations in ng/µL to calculate their pipetting volumes. Either stock can be supplied separately. When a reaction volume is provided, the required DNA volumes must fit within it; reserve space for buffer, ligase and other reagents.
Useful Checks
Use Primer Designer when cloning primers need restriction sites or overhangs before calculating ligation amounts.
Use Restriction Site Analyzer first to confirm enzyme sites and fragment sizes, then use this calculator for insert-to-vector molar ratio setup.
Minimum Inputs Needed
Vector length.
Insert length.
Vector DNA amount.
Insert:vector molar ratio.
Optional Settings
Vector / insert length units
Use bp or kb, depending on how your fragment size is written.
Custom insert:vector ratio
Select Custom and enter the insert-to-vector multiplier: enter 6 for a 6:1 molar ratio.
Reaction volume, µL (optional)
Enter this if you also want concentration estimates for the ligation mix.
Enter either or both measured stock concentrations to calculate pipetting volumes. These fields are separate from Vector DNA amount, which is a mass in ng.
How To Use
Enter vector length.
Enter insert length.
Enter vector mass in ng, not stock concentration in ng/microlitre.
Choose the insert:vector ratio.
Enter stock concentrations if you want pipetting volumes, and reaction volume if you want final DNA concentrations.
Click Calculate insert amount.
Understanding The Results
The main result is the insert DNA mass to add.
The formula is: insert mass = vector mass × insert length / vector length × insert:vector molar ratio.
The detailed result reports vector and insert femtomoles (fmol), total DNA mass and a comparison table. Femtomoles use the approximate dsDNA value of 660 g/mol per base pair.
The comparison table shows alternative ratios; the main result uses your selected ratio.
If reaction volume is entered, vector, insert, and total DNA concentration estimates are shown.
Copy result copies the full ligation setup for notes or protocols.
Accepted Input Formats
Lengths can be bp or kb.
Vector DNA amount is in ng.
Reaction volume is in µL.
Assumptions And Limitations
The calculator estimates DNA amounts and, when stock concentrations are supplied, the volumes to pipette. It does not assess DNA purity, end compatibility, phosphorylation or ligase performance. Follow your ligation protocol when choosing the ratio and reaction conditions.
Use it as an initial planning estimate and adjust based on your protocol.
Example
For a 3000 bp vector, 1000 bp insert, 50 ng vector, and 3:1 ratio, add 50 ng insert.
For that example, 10 ng/µL vector stock and 20 ng/µL insert stock give 5 µL vector and 2.5 µL insert. These illustrate the calculation, not recommended reaction conditions.
Changing calculation inputs clears the old result. Click Calculate insert amount to recompute it. Clear removes numeric inputs and results and restores 3:1, while keeping the selected length units.
Use this result with other tools
There is no direct send-to-tool button. Use Restriction Site Analyzer to select the intended digest fragments, then enter the retained vector and insert lengths here along with your vector mass and chosen molar ratio. Verify which fragment you are retaining rather than using the full undigested template length.
To see pipetting volumes, enter the vector and/or insert stock concentration in ng/µL. You can provide either stock separately. If you also enter a reaction volume, the DNA volumes must fit within it. Leave room for buffer, ligase and the other reagents. For overlap joining and primer design, use Assembly PCR Primer Designer or Gibson Assembly Primer Designer; those tools do not import a ligation calculation.
Use note: These tools are for research and educational planning. Check important calculations and sequence designs before ordering reagents or running experiments.