Bercovich J A, Grinstein S, Zorzopulos J
Laboratorio de Virologia, Hospital de Ninos R. Gutierrez, Buenos Aires, Argentina.
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.
