Ligation insert:vector ratio calculator
Set up a ligation by molecules, not by mass: with 50 ng of a 3000 bp vector, a 500 bp insert and a 3:1 ratio, you add 25 ng of insert. The picomoles of each fragment are shown too. Free in your browser.
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Run — free
Worked example
- Vector mass
- 50
- Vector length
- 3,000
- Insert length
- 500
- Molar ratio
- 3
- Length unit
- Bp
- Vector length bp
- 3,000
- Insert length bp
- 500
- Insert mass
- 25
A ligation insert-to-vector ratio calculator tells you how many nanograms of insert DNA to add to a known mass of linearized vector so the two fragments meet at a chosen molar ratio. Because a longer fragment is heavier per molecule, equal masses are not equal moles: a 3 kb plasmid out-masses a 500 bp insert roughly six to one, so a 1:1 ligation mixed by equal nanograms would starve the reaction of insert. The standard formula scales vector mass by the insert-to-vector length ratio and then by the target molar ratio, usually 3:1 for sticky ends or 1:1 to 5:1 when you are screening conditions.
Why equal nanograms are not a 1:1 ligation
Molecular cloning ligations join a linearized vector and an insert with T4 DNA ligase. The chemistry cares about colliding compatible ends, which means molar amounts, not the milligram-scale masses a spectrophotometer reports. Double-stranded DNA has an approximately constant mass per base pair, conventionally 650 grams per mole per base pair, so the number of moles in a given nanogram aliquot is inversely proportional to fragment length.
Fifty nanograms of a 3000 bp plasmid is therefore six times as many moles as fifty nanograms of a 500 bp insert. Mix those equal masses and you have built a 1:6 insert:vector reaction, the opposite of the 3:1 insert excess most sticky-end protocols recommend. The correction is a proportion: insert mass equals vector mass times (insert length divided by vector length) times the target molar insert-to-vector ratio.
For 50 ng of 3000 bp vector, a 500 bp insert, and a 3:1 ratio, that is 50 × (500 / 3000) × 3 = 25 ng. Blunt-end ligations often start nearer 1:1 or 5:1 and still use the same identity; only the ratio changes. This calculator applies that identity and also reports the implied picomoles using 650 g/mol per base pair so you can compare a NanoDrop reading with a molar protocol table without opening a spreadsheet.
Where a ligation insert:vector ratio calculator earns its keep
Teaching labs and cloning courses use a ligation insert-to-vector ratio calculator so students stop treating nanograms as interchangeable and can show the 25 ng insert that belongs with 50 ng of a 3 kb vector at 3:1. Core facilities and plasmid-prep notebooks keep the same identity next to spectrophotometer readings when a PCR product is shorter than the backbone and a 1:1 mass mix would under-insert the reaction. Failed validation does not consume balance.
Related searches for melting temperature of overlapping primers or Gibson assembly overlap design are different intents: stay on this page when the inputs are two fragment sizes plus a vector mass and the output is the insert nanograms for a target molar ratio.
What you can do with it
Sticky-end cloning prep
A bench scientist enters 50 ng of a 3000 bp backbone and a 500 bp PCR insert at 3:1 and pipettes the returned 25 ng of insert into the ligation.
FAQ
How do I calculate insert DNA mass for a ligation?
A 50 ng, 3000 bp vector with a 500 bp insert at 3:1 needs 25 ng of insert.
Why is a 3:1 insert:vector ratio the default?
Sticky-end T4 ligations usually start with a three-fold molar excess of insert so more insert ends find vector ends.
How much insert do I add to 50 ng of a 3000 bp vector at 3:1?
For a 500 bp insert you add 25 ng. The calculator multiplies 50 ng by 500 divided by 3000, then by the ratio of 3, so the insert outnumbers the vector three to one.
Why can't I mix insert and vector in equal nanograms?
Because a longer fragment weighs more per molecule. Fifty nanograms of a 3000 bp plasmid holds six times as many molecules as fifty nanograms of a 500 bp insert, so equal masses give a 1:6 reaction instead of 3:1.
Which ratio should I use for a blunt-end ligation?
Blunt-end ligations often start nearer 1:1 or 5:1, and you can screen ratios between those. The formula stays the same; only the ratio you enter changes.
Related tools
For developers — API access
Everything on this page is available programmatically. This section is for teams who want to wire it into their own systems; everyone else can just use the tool above.
API endpoint
Prefer to automate it? One authenticated POST creates the task; the result comes back by webhook or a signed link. The same capability also runs here on the web, by email and from Telegram — and soon from our app too.
Call it from your stack
curl -X POST https://api.kit.forhosting.com/bio/ligation-insert-vector-ratio \
-H "Authorization: Bearer $KIT_KEY" \
-H "Content-Type: application/json" \
-d '{"vector_mass":50,"vector_length":3000,"insert_length":500}'const res = await fetch("https://api.kit.forhosting.com/bio/ligation-insert-vector-ratio", {
method: "POST",
headers: {
"Authorization": `Bearer ${process.env.KIT_KEY}`,
"Content-Type": "application/json"
},
body: JSON.stringify({
"vector_mass": 50,
"vector_length": 3000,
"insert_length": 500
})
});
const { task_id } = await res.json();import os, requests
res = requests.post(
"https://api.kit.forhosting.com/bio/ligation-insert-vector-ratio",
headers={"Authorization": f"Bearer {os.environ['KIT_KEY']}"},
json={
"vector_mass": 50,
"vector_length": 3000,
"insert_length": 500
},
)
task_id = res.json()["task_id"]<?php
$res = file_get_contents("https://api.kit.forhosting.com/bio/ligation-insert-vector-ratio", false, stream_context_create([
"http" => [
"method" => "POST",
"header" => "Authorization: Bearer " . getenv("KIT_KEY") . "\r\nContent-Type: application/json",
"content" => '{"vector_mass":50,"vector_length":3000,"insert_length":500}',
],
]));
$task = json_decode($res, true);body := bytes.NewBufferString(`{"vector_mass":50,"vector_length":3000,"insert_length":500}`)
req, _ := http.NewRequest("POST", "https://api.kit.forhosting.com/bio/ligation-insert-vector-ratio", body)
req.Header.Set("Authorization", "Bearer "+os.Getenv("KIT_KEY"))
req.Header.Set("Content-Type", "application/json")
res, _ := http.DefaultClient.Do(req)Example request
{
"vector_mass": 50,
"vector_length": 3000,
"insert_length": 500
}Example response
{
"task_id": "tsk_a1b2c3d4e5f6a1b2c3d4e5f6",
"type": "bio.ligation_insert_vector_ratio",
"status": "queued",
"_links": {
"result": "/tasks/tsk_…/result"
}
}The API is asynchronous: the call returns a task_id immediately and the result arrives by webhook. Polling is capped at 1 req/s per task.
Pricing
Published price — no tokens, no invented credits. A failed task is never charged.
Limits
max_mass_ng | 1000000000000 |
max_length_bp | 1000000000 |
Errors
| HTTP | Code | Meaning |
|---|---|---|
401 | unauthorized | Missing or invalid API key. |
402 | insufficient_balance | Your balance doesn't cover the task price. |
404 | unknown_type | That task type doesn't exist. |
429 | rate_limited | Too many requests. Use the webhook instead of polling. |
Technical notes
Input contract, response shape, and error behaviour
POST a JSON body with three required numbers. vector_mass is the nanograms of linearized vector you will actually pipette (aliases vector_ng, ng_vector, vector). vector_length and insert_length are the two fragment sizes after any restriction digest or PCR clean-up; they share length_unit, an enum of bp or kb that defaults to base pairs (aliases unit, size_unit). Optional molar_ratio is the insert:vector molar target and defaults to 3, the usual sticky-end starting point; aliases are ratio, insert_vector_ratio, and iv_ratio. Each numeric field may arrive as a JSON number or a decimal or scientific string. Missing required fields, empty strings, non-finite values, zeros, negatives, and values outside the published limits fail with invalid_input that names the field. Those failures are not billed. On success the body echoes the inputs, reports vector_length_bp and insert_length_bp after the optional kilobase conversion, returns insert_mass in nanograms, mass_ratio as insert_mass divided by vector_mass, and ratio_label such as 3:1. vector_pmol and insert_pmol use 650 g/mol/bp, so a 50 ng, 3000 bp vector is about 0.0256 pmol and a 3:1 insert is about 0.0769 pmol. formula restates insert_mass = vector_mass × (insert_length / vector_length) × molar_ratio; formula_applied substitutes this call's numbers. Pair the mass with your own ligase-buffer table; this endpoint does not choose enzyme units or incubation time.
Where a ligation insert:vector ratio calculator earns its keep
Keep masses and lengths inside the declared ceilings, pin insert_mass and formula_applied in tests, and you get reproducible 25-nanogram answers for the textbook 50 ng / 3000 bp / 500 bp / 3:1 case across a lab notebook, an LMS plugin, and a cloning pipeline.
Integration use cases
Classroom molar-ratio worksheets
Students check that equal nanograms are not a 1:1 ligation and compare insert_mass against a hand-worked 50 × (L_insert / L_vector) × ratio line.
Cloning-script and LMS oracles
Automation pins insert_mass for the textbook 3:1 case and invalid_input when a fragment length is missing or non-positive.
Technical questions
Can I enter fragment sizes in kb instead of bp?
Yes. Set length_unit to kb (both lengths share that unit). 3 kb and 0.5 kb at the same mass and ratio give the same insert_mass as 3000 bp and 500 bp, because the scale cancels in the length ratio.
Does the calculator assume double-stranded DNA?
Yes. insert_mass uses the length ratio, which is independent of the mass-per-base-pair constant. vector_pmol and insert_pmol use the conventional 650 g/mol/bp average for dsDNA.
What does the ligation insert:vector ratio calculator cost?
The browser tool is free. API requests use prepaid balance at $0.002 per successful call; invalid input is not billed.