Salvaging recombinants from low-efficiency ligase reactions for more efficient subcloning.
Certain types of ligase reactions can be problematic, such as those involving PCR products, blunt-ends and multiple DNA inserts. A simple PCR-based strategy was developed to overcome cloning difficulties with these inefficient ligase reactions. After an initial ligase reaction, primers complementary to the vector are utilized to amplify the DNA fragment from (the few) successful recombinants in the ligation mixture. This DNA fragment is processed for use in a more conventional and straightforward ligase reaction. We demonstrate the potential of the technique by applying it to a variety of difficult ligase reactions.
Sun
H W
HW
Yale University School of Medicine, New Haven, CT 06510, USA.
Lolis
E
E
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics*; D008025:Ligases / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017467:Receptors, Erythropoietin / Q000235:genetics
D017931:DNA Primers; D004274:DNA, Recombinant; D017467:Receptors, Erythropoietin; D008025:Ligases