Ligation Calculator
Estimate the insert DNA mass for your ligation mix
Enter insert/vector sizes, vector mass, and a molar ratio to get the recommended insert mass.
DNA & ratio
Introduction / overview
This ligation calculator estimates the insert DNA mass you should add to a ligation reaction, based on: vector mass, insert length, vector length, and your chosen insert:vector molar ratio.
💡 Why this is useful: cloning often fails because the insert is underrepresented. A clear molar-ratio target helps you set up reactions consistently across different plasmid sizes.
Who typically uses this?
- Students learning cloning for the first time
- Bench scientists running routine restriction/ligation cloning
- Anyone troubleshooting “no colonies” and wanting a quick ratio sanity check
Tip: if your limiting factor is concentration (not mass), pair this with our DNA Concentration Calculator to convert between ng/µL and total ng.
How to use the ligation calculator
Enter your insert length
Use bp or kb — the calculator converts for you.
Enter your vector length
Again, bp and kb are both fine.
Enter your vector DNA mass
Use ng, µg, mg, or g. (Most ligations are in the ng–µg range.)
Choose the insert:vector molar ratio
A common starting point is 3:1. Larger ratios can help when the insert is small or ligation efficiency is low.
Check the blue (auto-calculated) field
The calculator highlights one field in blue — that is the current auto-calculated result. By default it solves for Insert DNA mass (shown as an input field under the molar ratio).
You can also reverse-calculate. Just directly edit the blue result field (for example, type an insert mass you want to use), and the calculator will automatically switch the result to another field and solve for it. The molar ratio dropdown stays as a fixed input and is not part of the reverse-solving.
Unit conversions (quick refresher)
- 1,000 bp = 1 kb
- 1 bp = 0.001 kb
- 1 µg = 1,000 ng
Many labs prefer not to go too low on DNA mass (for example, targeting tens of ng or more) just to reduce pipetting error. If the blue (auto-calculated) mass looks tiny, consider increasing the vector mass, concentrating DNA, or using a slightly higher ratio.
Example 1 (quick)
Insert = 1.2 kb, Vector = 3.6 kb, Vector mass = 50 ng, Ratio = 3:1
Insert mass = (50 × 1.2) / 3.6 × 3 = 50 ng
Example 2 (bp input + different ratio)
Insert = 500 bp (0.5 kb), Vector = 5 kb, Vector mass = 100 ng, Ratio = 5:1
Insert mass = (100 × 0.5) / 5 × 5 = 50 ng
How to calculate the molar ratio
For double-stranded DNA, mass scales roughly with length (bp). That’s why a simple length-based correction works well for planning ligations. This calculator uses:
Vector/insert planning formula
Required insert mass = (Vector mass × Insert length) / Vector length × Molar ratio
Lengths can be entered in bp or kb (they’re converted internally). Mass can be entered in ng/µg/mg/g and is converted as needed. In bidirectional mode, the calculator can also rearrange this relationship to solve for a different variable.
What the variables mean
- Vector mass: how much backbone DNA you plan to use (e.g., 20–100 ng)
- Insert length: size of the fragment you’re cloning
- Vector length: size of the linearized plasmid backbone
- Molar ratio: your target insert:vector molecule ratio (often 3:1)
The ratio you choose depends on insert size, vector size, and how clean your DNA is. As a rule of thumb, when the vector is much larger than the insert, a higher insert:vector ratio can help.
Ligation reaction (what’s happening)
In a ligation, you’re trying to create a recombinant DNA molecule by forming a covalent bond between DNA ends. A common workhorse enzyme is T4 DNA ligase, which uses ATP as an energy source.
Sticky ends
Many restriction enzymes cut DNA unevenly, leaving short overhangs. Compatible overhangs can pair up, making ligation easier.
Vector vs. insert
In cloning language, the vector is your backbone (plasmid), and the insert is the fragment you want to add.
✅ A simple mental model: Vector = backbone + insert. Your goal is to give the reaction enough insert molecules to find vector ends.
If you’re tuning conditions (temperature, time, enzyme amount), the calculator won’t replace a protocol — but it does remove one big source of variability: the DNA ratio.
Real-world examples / use cases
Here are a few common situations where a fast insert-mass estimate is genuinely helpful.
Routine restriction/ligation cloning
Plug in your fragment and plasmid sizes, pick 3:1, and get a starting insert mass in seconds.
Small insert, big vector
When the insert is short, you often need less mass for the same molar count — the calculator keeps that intuitive.
Troubleshooting low colony counts
Try comparing 2:1 vs 3:1 vs 5:1 ratios to see what insert mass change you’re actually making.
Adapter / linker ligations
Useful any time you need a predictable excess of a shorter DNA piece relative to a longer one.
Planning pipetting volumes
After you have the target ng, convert it to µL using your DNA concentration. Use the DNA Concentration Calculator.
Tips, FAQs, limitations
Common scenarios (and when this helps most)
- You know vector mass, but you’re unsure how much insert to add.
- You changed to a different plasmid size and want the same molar ratio as before.
- You’re comparing multiple ratios (1:1, 2:1, 3:1, 5:1, 7:1) to optimize.
When it may be less applicable: reactions dominated by factors like DNA end compatibility, enzyme freshness, or impurities. In those cases, the ratio is still worth checking — it’s just not the only variable.
Start with 3:1
If you’re unsure, 3:1 is a practical default. Then test 2:1 or 5:1 as quick variants.
Avoid tiny pipetting
If the insert mass converts to an impractically small volume, consider concentrating DNA or increasing the vector mass.
Use correct lengths
Use the final ligated insert length and the linearized vector backbone length (in bp or kb).
Check DNA concentration
If you only know ng/µL, convert it first using our DNA Concentration Calculator.
Frequently Asked Questions
Do I always need a 3:1 insert:vector ratio?
No — it’s a solid starting point, not a law. If you’re getting background colonies or self-ligation, you might lower it; if you’re struggling to get colonies, you might try a higher ratio.
Why does insert length affect the required mass?
Because molar ratio is about molecule counts, not ng. A longer fragment weighs more per molecule, so you need more ng to get the same number of molecules.
What units should I use?
Any supported units are fine. The calculator converts internally so the math is consistent. Just make sure you’re entering lengths for the same DNA species (insert fragment vs vector backbone).
My computed mass seems too small. Is that wrong?
Not necessarily — depending on your lengths and ratio, the calculated mass can be surprisingly small. Practically, tiny volumes can be error-prone, so many labs prefer to adjust the setup to pipette comfortably.
Does this replace a ligation protocol?
No. It only targets the DNA ratio piece. Enzyme activity, buffer, temperature, incubation time, end compatibility, and DNA purity still matter a lot.
Limitations / disclaimers
- This tool uses a simple length-based model for dsDNA to solve for one missing variable (the blue field).
- It does not account for DNA end compatibility, ligase efficiency, DNA damage, or contaminants.
- For lab work, always follow your institution’s safety rules and a validated protocol.
External references / sources
Related Calculators
Cat Pregnancy Calculator
The cat pregnancy calculator will compute the due date of your favorite feline.
Dog Life Expectancy Calculator
The dog life expectancy calculator estimates the age of your dog based on their breed and gives their average life expectancy.
Dog Age Calculator
Convert your dog's age to human years based on breed and size.
Dog Pregnancy Calculator
The dog pregnancy calculator provides you with your dog's due date.
Benadryl Dosage Calculator for Dogs
Our Benadryl dosage calculator for dogs will compute an optimal dose of Benadryl (Diphenhydramine) for your pet.
Dog Quality of Life Calculator
Discover how to measure a dog's quality of life with our insightful pet quality of life scale calculator. Ensure their well-being now!