What it does
Ligations and assemblies work best when the pieces are mixed by number of molecules, not by weight. A 1,200 bp insert weighs less per molecule than a 5,400 bp vector, so a 3:1 molar ratio is not 3:1 in ng. This page does the conversion for any number of fragments and turns it into a pipetting list:
- Amounts. For each insert: fmol = vector fmol × ratio, then ng from the length. The vector’s amount sets everything.
- Volumes. µL of each fragment from its concentration, the buffer or master mix volume (total ÷ strength), the enzyme volume, and the water that fills the rest.
- Checks. Whether the DNA fits in the room left, the most vector that would fit, and which fragments need less than 0.5 µL (hard to pipette accurately), with a dilution that brings them to at least 1 µL.
- Other ratios. The first insert’s ng and µL at 1:1, 2:1, 3:1, 5:1, 7:1 and 10:1.
How to use it
- Reaction. Pick Ligation (10× buffer plus 1 µL ligase in 20 µL) or Assembly (2× master mix, enzyme included), or type your own total volume, buffer strength and enzyme volume.
- Vector amount. How many ng of vector you want in the reaction.
- Fragments. One row each: name, length in bp, concentration in ng/µL and molar ratio to the vector. The first row is the vector.
- Pipette. Follow the table from the top: buffer, water, DNA, and the enzyme last.
Worked example
The made-up example assembles a 5,400 bp vector (62.5 ng/µL) with three pieces: Insert A, 1,200 bp at 40 ng/µL, 3:1; Insert B, 850 bp at 25 ng/µL, 3:1; and Linker C, 400 bp at 18 ng/µL, 5:1. With 50 ng of vector, that is 15 fmol of vector, so each 3:1 insert is 45 fmol. Insert A comes to 33.3 ng (0.83 µL), the same as the shortcut 50 × 3 × 1,200 ÷ 5,400. In a 20 µL ligation with 2 µL of buffer and 1 µL of ligase, the DNA takes 3.61 µL and water makes up 13.4 µL. Up to 236 ng of vector would still fit at these ratios.
Switch to Assembly and the 2× master mix takes 10 µL, leaving 6.39 µL of water.
Good to know
- Ratios of 1:1 to 10:1 are commonly tried; very short inserts are often added in larger excess. Your kit’s manual has its own advice.
- Measure concentrations after clean-up; leftover primers and nucleotides inflate A260.
- Molar amounts assume linear double-stranded DNA, which is what you ligate or assemble.
FAQ
Why fmol and not pmol? Typical vector amounts are tens of fmol (hundredths of a pmol); both are shown in the download.
What if the DNA does not fit? Use less vector (the tool gives the most that fits), concentrate the DNA, or raise the total volume.
Is anything uploaded? No. Everything runs in your browser.
Where the numbers come from
- Molar ratio arithmetic: pmol = ng ÷ molar mass × 1000, with the mass of linear double-stranded DNA worked out by fibub from the nucleotide formulas (the same as the DNA concentration tool). Written by fibub.
- The 3:1 starting ratio, 10× ligase buffer and 2× assembly master mix are common lab conventions, used as editable defaults; no kit manual text or tables are copied. Follow your kit's instructions where they differ.
- Checked in the tests against an independent Python calculation. The example fragments are made up.
Standard scientific values, such as constants, atomic weights and melting points, are facts found in every textbook; we name where ours were checked. If you are an author or publisher and want a value, a credit or a table changed or removed, tell us and we will act quickly.
Last updated 2026-11-09.