An efficient method for blunt-end ligation of PCR products.
This report presents data demonstrating a simple method that can potentially be extended to a wide range of cloning strategies to increase the yield of insert-containing recombinants. The method requires that the ligation of an insert to a vector does not regenerate the original restriction enzyme recognition sequence. In the example presented, PCR products were blunt-end ligated to a SmaI-cut vector, in the presence of SmaI endonuclease. The addition of the restriction enzyme to the ligation reaction dramatically favored the ligation of insert to vector rather than vector self-ligation.
Liu
Z G
ZG
Molecular and Cellular Biology Program, Morrill Science Center, University of Massachusetts, Amherst 01003.
Schwartz
L M
LM
D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D010957:Plasmids; D016133:Polymerase Chain Reaction; D014169:Transformation, Bacterial
D004247:DNA; C025651:CCCGGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific