DNA Ligation Calculator
Plan insert mass, compare molar ratios and balance a reaction worksheet.
DNA & reaction inputs
Insert mass & molar balance
Recombinant plasmid preview
Compare insert : vector ratios
| Ratio | Insert mass | Insert volume |
|---|---|---|
| 1:1 | 16.67 ng | 0.67 µL |
| 2:1 | 33.33 ng | 1.33 µL |
| 3:1 | 50.00 ng | 2.00 µL |
| 5:1 | 83.33 ng | 3.33 µL |
| 7:1 | 116.67 ng | 4.67 µL |
| 10:1 | 166.67 ng | 6.67 µL |
Highlighted row matches the selected molar ratio.
Reaction worksheet
| Component | Volume |
|---|---|
| Vector DNA | 1.00 µL |
| Insert DNA | 2.00 µL |
| 10× buffer | 2.00 µL |
| Ligase · user supplied | 1.00 µL |
| Nuclease-free water | 14.00 µL |
| Total | 20.00 µL |
Arithmetic worksheet, not a validated protocol.
TOPO uses a different chemistry. T4 buffer and enzyme rows apply only to the T4 worksheet. No incubation, transformation or product-performance protocol is inferred.
Formula & example
Formula
Insert mass = vector mass × (insert bp / vector bp) × molar ratio
Example calculation
50 × (1,000 / 3,000) × 3 = 50 ng
Units and calculation assumptions
The model assumes double-stranded DNA with the same average mass per base pair in vector and insert. The length ratio therefore converts a molar ratio into a mass ratio. Optional fmol values use approximately 660 g/mol per bp. A nominal single-insert construct length is vector bp + insert bp; it does not account for sequence edits, compatibility, background products or reaction success.
DNA volume (µL) = mass (ng) ÷ stock concentration (ng/µL). For the T4 worksheet, buffer volume = total volume ÷ stock multiplier (for a modeled 1× final concentration); water is the remaining volume after all user-entered components. No negative water volume is accepted. TOPO mode reports only DNA volumes and unallocated kit volume.
Frequently asked questions
Why use a molar ratio instead of a mass ratio?
The number of DNA molecules depends on fragment length as well as mass. Equal masses of a short insert and a long vector contain different molecule counts. A molar ratio compares molecule counts.
Does TA cloning use the same reagents as TOPO?
No. Conventional TA ligation can use T4 DNA ligase, while TOPO uses topoisomerase-based chemistry. Select TOPO for a DNA-volume check and follow the kit manual for its remaining components.
What if the calculated volumes exceed the reaction?
The worksheet is invalid and no water amount is supplied. Review the entered stock concentrations, DNA amounts and total reaction volume. The independently calculated insert mass remains visible.
Can this diagram predict ligation success?
No. It is a schematic of a nominal vector-plus-insert construct. End compatibility, sequence, purity, chemistry and experimental conditions are not assessed.
What molar ratio should I use?
The examples provide starting arithmetic comparisons, not a universal protocol. NEB describes ratios that depend on relative fragment sizes and cloning context. Refer to your validated method or product instructions.
What concentration should the reaction be?
This calculator does not set an optimal reaction concentration. Optional stock concentrations convert DNA masses to pipetting volumes; enzyme and kit requirements remain product specific.
Sources & Reference Guides
New England BiolabsInsert : vector molar-ratio calculation.Thermo FisherSCIENTIFICThermo Fisher ScientificTA and TOPO chemistry explained.
NCBI · NIH / NLMRecombinant DNA background.Independent references, not institutional endorsement or validation. Thermo Fisher name is a text reference, not an official logo.
Plan insert mass, compare molar ratios and balance a reaction worksheet.