CH 5 · notes
Assumptions
Ligation calculator for one insert and one vector: enter both lengths in base pairs, the vector mass in nanograms, and the insert:vector molar ratio. The page returns the insert mass and both amounts in picomoles and femtomoles. An optional insert stock, in ng/µL, adds the volume that contains that insert mass. Leave the stock blank and the volume stays off.
Double-stranded DNA only. One base pair is 650 grams per mole, with no end correction. That is the average written in the NEB PCR cloning ligation protocol, and it is the convention this site uses for dsDNA mass and moles. A calculator that adds an end correction will not match these picomoles, especially for a short fragment. Single-stranded DNA and RNA are a different mass per residue, so they are not accepted here.
The ratio is moles of insert divided by moles of vector. The preset 3:1 is three moles of insert for one mole of vector. 1:1 and 5:1 are the other presets. Custom is any number above zero. The mass fields start empty. Choosing a preset before you type does not invent a vector mass, and it does not fill in 50 ng for you.
How it works
insert ng = vector ng × ratio × insert bp ÷ vector bp
pmol = ng × 1000 ÷ (bp × 650)
650 g/mol per bp, dsDNA, no end correction
Because every base pair has the same average mass, the molecular weight is length times 650. The insert mass is then the vector mass times the ratio times the insert length, divided by the vector length. The same 650 converts each mass into picomoles: nanograms times 1000, divided by length times 650. Femtomoles are those picomoles times 1000. Doing the mole conversion and then converting back to nanograms gives the same insert mass. The page checks that the two routes agree before it shows a number.
Display is four significant figures, half away from zero, the same rule as the solution channels. The WORKING lines quote those strings. They do not round to a nearby whole number of nanograms. Prep steps quote the same strings: add the vector mass you typed, add the calculated insert mass, and, only when a stock is filled in, pipette that volume of the stock. They do not name a ligase, a buffer, or an incubation. When a stock is filled in, the insert volume is compared with the minimum pipette volume you enter. The comparison is strict: a smaller volume lights the amber pipette mark and tells you to dilute the insert stock. An equal volume is not marked. A blank stock has no volume, so it is not compared.
Copy result is only the insert mass, a number, a space, and ng. Copy protocol is the longer text: both lengths, the ratio, both amounts, the stock and volume when you entered a stock, the numbered steps, and the working lines. Print uses the site print stylesheet. The footer under the steps is molicheck.com/ligation.
Worked example
Vector length 3000 bp, insert length 1000 bp, vector mass 50 ng, ratio 3, insert stock 100 ng/µL. Minimum pipette volume 1 µL. The insert mass is 50.00 ng. The vector is 0.02564 pmol (25.64 fmol). The insert is 0.07692 pmol (76.92 fmol). The stock volume is 0.5000 µL. That volume is below the minimum, so the page shows: Below minimum pipette volume. Dilute the insert stock.
- insert ng = 50 ng × 3 × 1000 bp ÷ 3000 bp = 50.00 ng
- vector pmol = 50 ng × 1000 ÷ (3000 bp × 650 g/mol) = 0.02564 pmol
- vector fmol = 25.64 fmol
- insert pmol = 3 × vector pmol = 0.07692 pmol
- insert fmol = 76.92 fmol
- insert volume = 50.00 ng ÷ 100 ng/µL = 0.5000 µL
- Add 50 ng of vector DNA (0.02564 pmol, 25.64 fmol).
- Add 50.00 ng of insert DNA (0.07692 pmol, 76.92 fmol).
- Pipette 0.5000 µL of the 100 ng/µL insert stock.
Common mistakes
Multiplying the vector mass by three and stopping there is right only when the insert and the vector are the same length. A 1000 bp insert against a 3000 bp vector at 3:1 is the same mass as the vector, not three times that mass. Swap the two lengths and the insert mass jumps.
A ratio written as vector:insert instead of insert:vector runs the equation backwards. On this page the first number is the insert. 3:1 is not one part insert to three parts vector.
Leaving the stock at zero is not the same as leaving it blank. Zero is rejected. Blank omits the volume and still shows the mass. A stock in ng/µL is not a molar concentration. The page does not convert mg/mL or nM into that field.
FAQ
What does 3:1 mean?
Three moles of insert for one mole of vector. The default preset is 3:1. 1:1 and 5:1 are the other presets. Custom accepts any ratio above zero.
Why is the insert mass not just the vector mass times three?
Only when the two fragments are the same length. A shorter insert weighs less per mole, so the mass is vector mass times the ratio times insert length divided by vector length.
Does this page handle single-stranded DNA or RNA?
No. This page is double-stranded DNA only. One base pair is 650 g/mol, with no end correction. Single-stranded DNA and RNA are not accepted.
Do I have to enter an insert stock concentration?
No. Leave it blank and the page still gives the insert mass and both amounts in pmol and fmol. A filled stock adds the microlitre volume, which is that mass divided by the stock.
When does the pipette warning appear?
Only when an insert stock is filled in and that insert volume is strictly less than the minimum pipette volume you entered. An equal volume is not marked. The message is Below minimum pipette volume. Dilute the insert stock. Leave the stock blank and there is no volume to compare.
Sources
- NEB. Ligation protocol for the PCR cloning kit (E1202). States 650 daltons per base pair, and insert nanograms equal to the ratio times vector nanograms times insert length over vector length. This page uses that figure as 650 g/mol per base pair of double-stranded DNA, with no end correction.
- NEB. Tips for maximizing ligation efficiencies. Recommends about 20–30 fmol of vector and insert:vector ratios from 1:1 to 1:10 for a single insert. This page does not choose a reaction volume or an enzyme amount from that note.
Open the home index for the other calculators. Primer Tm is a melting temperature, not a ligation ratio. C1V1 is one dilution step. Neither page uses this 650 g/mol line.