Effect of DNA concentration on recombinant plasmid recovery after blunt-end ligation.
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.
Bercovich
J A
JA
Laboratorio de Virologia, Hospital de Ninos R. Gutierrez, Buenos Aires, Argentina.
Grinstein
S
S
Zorzopulos
J
J
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003001:Cloning, Molecular / Q000379:methods*; D011088:DNA Ligases / Q000378:metabolism*; D004274:DNA, Recombinant / Q000302:isolation & purification* / Q000378:metabolism; D010957:Plasmids
D004274:DNA, Recombinant; D011088:DNA Ligases