An optimized recipe for cloning of the polymerase chain reaction-amplified DNA inserts into plasmid vectors.
This study compares a number of parameters that are important in the ligation of the polymerase chain reaction-amplified DNA inserts into plasmid vectors and their efficient transformation to bacterial cells. The parameters covered were: T4 polynucleotide kinase treatment followed by either the large fragment of E. coli DNA polymerase or T4 DNA polymerase reactions, the amount of T4 DNA ligase, temperature and duration of ligation, molar ratio of insert to vector as well as the total DNA concentration. The results show that the T4 polynucleotide kinase-treated group without further enzymatic manipulation, at an insert to vector ratio of 3:1 gave the highest recombination efficiency when 10 microg/ml DNA and 20 units T4 DNA ligase were applied for ligation for 12 h at 4 degrees C.
Topcu
Z
Z
Department of Pharmaceutical Biotechnology, School of Pharmacy, Ege University, Izmir, Turkey. ztopcu@bornova.ege.edu.tr
D000818:Animals; D017122:Bacteriophage T4 / Q000201:enzymology; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular / Q000379:methods*; D011088:DNA Ligases; D017931:DNA Primers / Q000235:genetics; D004272:DNA, Mitochondrial / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D066298:In Vitro Techniques; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D014170:Transformation, Genetic
D017931:DNA Primers; D004272:DNA, Mitochondrial; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D004259:DNA-Directed DNA Polymerase; D011088:DNA Ligases