Ligation Calculator: Insert Mass and Molar Ratio for DNA Cloning

Your details

Sticky-end ligations are more efficient; blunt-end reactions typically need a higher insert-to-vector ratio.
Amount of linearised, dephosphorylated vector DNA in nanograms. Typically 50-100 ng per reaction.
ng
Length of the linearised vector in base pairs (e.g. pUC19 is 2686 bp).
bp
Length of the insert fragment in base pairs.
bp
Number of insert molecules per vector molecule. For sticky-end ligations, 3:1 is standard. Blunt-end ligations often benefit from 5:1 or higher.
Total ligation reaction volume. T4 DNA Ligase reactions are typically 10-20 µL.
µL
Combined volume of 10x ligation buffer and T4 DNA Ligase (e.g. 2 µL buffer + 1 µL ligase = 3 µL). The remaining volume is filled with water.
µL
Concentration of your vector stock. Used to calculate the volume of vector to add.
ng/µL
Concentration of your insert stock. Used to calculate the volume of insert to add.
ng/µL
Insert mass requiredGood reaction setup
50ng

Mass of insert DNA to add to the reaction

Vector volume5µL
Insert volume10µL
Water to add2µL
Vector amount25.25fmol
Insert amount75.76fmol
Actual molar ratio3
Vector5
Insert10
Water2

Ligation ready: 50.0 ng insert at 3.0:1 molar ratio.

  • You need 50.0 ng of insert DNA (10.00 µL of your stock) for this reaction.
  • The reaction uses 5.00 µL vector + 10.00 µL insert, confirming a 3.0:1 insert-to-vector molar ratio.

Next stepIncubate your T4 DNA Ligase reaction at 16 degrees C for 1-16 hours (or use a quick-ligation kit at 25 degrees C for 15 minutes), then transform 1-5 µL directly into competent cells.

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