Molecular cloning of PCR fragments with cohesive ends.
Use of the polymerase chain reaction (PCR) provides a convenient means of generating DNA fragments for insertion into plasmids. Large quantities of the desired insert, bounded by convenient restriction sites, may be synthesized. The primers are chosen to span a known region of interest, and extended at their 5'-ends to include the desired restriction sites. Amplification of the target sequence is followed by precipitation of the product with ammonium acetate and ethanol to remove the primers. A small amount of product is analyzed by gel electrophoresis to ensure correct amplification, the remainder is digested with the appropriate restriction enzyme(s). Restricted insert DNA is added to similarly restricted plasmid DNA in several ratios and incubated with DNA ligase to recircularize. Ligation products are used to transform competent bacteria. Clones containing inserts are identified by restriction digestion of plasmid minipreps from bacterial colonies.
Delidow
B C
BC
Department of Biochemistry and Molecular Biology, Marshall University School of Medicine, Huntington, WV 25704, USA. delidow@marshall.edu
D003001:Cloning, Molecular / Q000379:methods*; D053938:DNA Fragmentation; D004926:Escherichia coli / Q000235:genetics; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D014170:Transformation, Genetic