A simple two-step method for efficient blunt-end ligation of DNA fragments.
The formation of recombinant plasmids results from ligation between one end of the linearized vector and one end of the insert (favored by high DNA concentration), followed by self-ligation of the newly created hybrid molecule (favored by low DNA concentration). Standard protocols recommend an average DNA concentration at which both events may occur. Since this DNA concentration is not optimum for both ligation events, efficient blunt-end ligation is compromised. We describe a method for blunt-end ligation starting at a high DNA concentration for 1 h then at 1/20 the initial DNA concentration overnight. The number of recombinant plasmids obtained with this method is about 10-fold higher than with standard protocols. Restriction digestion and agarose gel electrophoresis of 10 recombinant plasmids obtained with the two-step ligation method showed that all plasmids contained one copy of the insert.
Damak
S
S
Centre for Molecular Biology, Lincoln University, Canterbury, New Zealand.
Bullock
D W
DW
D001709:Biotechnology / Q000379:methods*; D011088:DNA Ligases / Q000378:metabolism; D004256:DNA Polymerase I / Q000378:metabolism; D004274:DNA, Recombinant; D015246:Deoxyribonuclease EcoRI; D004926:Escherichia coli / Q000235:genetics; D019204:GTP-Binding Proteins / Q000235:genetics; D005822:Genetic Vectors; D010957:Plasmids; D014169:Transformation, Bacterial; D020668:rap GTP-Binding Proteins
D004274:DNA, Recombinant; D004256:DNA Polymerase I; D015246:Deoxyribonuclease EcoRI; D019204:GTP-Binding Proteins; D020668:rap GTP-Binding Proteins; D011088:DNA Ligases