Efficient DNA subcloning through selective restriction endonuclease digestion.
Described here is a selective restriction endonuclease digestion method that eliminates the electrophoresis step that is usually used during the subcloning of new DNA sequences into typical E. coli-based plasmids. The method increases yield while decreasing laboratory resource and time utilization. By using donor and acceptor sequences that contain unique restriction sites found only outside of the intended recombination sequences, the initial digestion products can be directly combined without electrophoresis if the ligation step is followed by a selective digestion using the unique restriction enzymes before transformation. This system is based on the several order of magnitude decrease in transformation efficiency of linearized compared to circular plasmids. As an example, this method was used to obtain recombinants between a 3.6 kb acceptor plasmid and 3.0 kb insert following one ligation reaction after the failure of nine standard reactions using similar amounts of input DNA. It is particularly applicable to situations in which low subcloning efficiencies are expected. The technique can be extended to a large percentage of planned recombinations by using nonidentical compatible cohesive or blunt-ended fragments, or site-directed mutagenesis.
Spear
M A
MA
University of California at San Diego, USA.mspear@ucsd.edu
D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000235:genetics; D011088:DNA Ligases / Q000737:chemistry; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004586:Electrophoresis; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D011995:Recombination, Genetic; D015183:Restriction Mapping / Q000379:methods*; D013379:Substrate Specificity / Q000235:genetics*; D014169:Transformation, Bacterial / Q000235:genetics
D004247:DNA; D004262:DNA Restriction Enzymes; D011088:DNA Ligases