How to use it
- Enter the vector's length and concentration, and how much of it goes into the reaction.
- Choose the assembly. One insert defaults to 3 : 1; several fragments default to equimolar.
- Paste the inserts, one per line. Add a fourth cell to give a line its own molar ratio.
- Check the reaction volume, buffer strength and ligase volume. The defaults are 20 µL, 10× and 1 µL.
- Pipette in the order of the table. Water is the largest volume so it goes first, and the ligase goes last.
The formulas, and what to watch for
Matching the molar ratio is one division. The rest is filling the tube.
Mass to moles
A base pair has an average mass, so a length and a mass give an amount. The same average is used as in this site's ng ↔ pmol converter.
- pmol
- ng × 1000 / (bp × 650)
- mass of 1 pmol
- bp × 650 / 1000 ng
How much insert
The insert's amount is the vector's amount times the molar ratio, converted back into a mass and then a volume.
- insert pmol
- vector pmol × ratio
- insert ng
- insert pmol × length × 650 / 1000
- insert µL
- insert ng ÷ concentration
Filling the tube
The buffer volume is derived rather than looked up: a 10× buffer becomes 1× at one part in ten.
- buffer µL
- reaction volume ÷ buffer strength
- water µL
- reaction − DNA − buffer − ligase
What to watch for
- When the water comes out negative nothing is shown but the size of the overshoot. It happens with dilute DNA, and putting in less vector is the easiest fix.
- 3 : 1 is the starting point for standard cloning where the insert is smaller than the vector (Addgene). Large inserts and blunt ends usually mean trying a range of ratios in parallel.
- Multi-fragment assemblies start equimolar. Addgene notes that some labs see a sharp drop in success beyond five fragments in one reaction.
- 650 g/mol per base pair is an average. Short fragments depart from it, and that error goes straight into the molar ratio.
- Add the ligase last and do not vortex. It is in glycerol and will layer; mix gently with the pipette.
- This calculates what goes in, and stops there. Temperature, time and blunt-end handling come from your enzyme's manual.
Frequently asked questions
Three insert to one vector. It is a molar ratio, not a mass ratio, so an insert shorter than the vector goes in at fewer ng than the vector does. The pmol column shows the ratio directly.
The DNA volumes already exceed the reaction volume. Putting in less vector is the easiest fix and usually enough; otherwise scale the reaction up or concentrate the DNA.
Switch the assembly to several inserts and list them one per line. They default to equimolar; a fourth cell on a line gives that fragment its own ratio.
Put 5 in the buffer strength field. A 20 µL reaction then takes 4 µL — the volume is calculated from the strength rather than looked up.