Effect of DNA concentration on recombinant plasmid recovery after blunt-end ligation

Biotechniques. 1992 Feb;12(2):190, 192-3.


We describe conditions for optimal recovery of recombinant plasmids after blunt-end ligation. It was found that one of the most critical parameters of the blunt-end ligation reaction is total DNA concentration (vector plus incoming DNA). This concentration was optimal in the range of 1-5 micrograms/ml of reaction mixture. Concentrations larger than 10 micrograms/ml result in strong inhibition. The optimal molar relationship between incoming DNA and vector was found to be 1 or less. Under these conditions, using dephosphorylated vector, recombinants are generated at a frequency of 10(6) colonies per microgram of insert, provided that transforming efficiency is about 5 x 10(7) colonies per microgram of plasmid DNA.

Publication types

  • Comparative Study

MeSH terms

  • Chemical Phenomena
  • Chemistry, Physical
  • Cloning, Molecular / methods*
  • DNA Ligases / metabolism*
  • DNA, Recombinant / isolation & purification*
  • DNA, Recombinant / metabolism
  • Plasmids*


  • DNA, Recombinant
  • DNA Ligases