Comparison of Two Gel Electrophoresis-based DNA Micropurification Methods for Molecular Cloning

• Published in: Biotecnología aplicada Vol. 18; no. 1; pp. 29 - 31
• Main Authors: Hardy, Eugenio, Silva, Ricardo, Pupo, Elder, Hermida, Lisset, Coizeau, Edelgis, Niebla, Olivia, Castellanos-Serra, Lila
• Format: Journal Article
• Language: English
• Published: Cuba Elfos Scientiae 31.12.2001
• Subjects: bromuro de etidio; clonación; cloning; electroforesis en gel; ethidium bromide; gel electrophoresis; luz ultravioleta; polyacrylamide; tinción con zinc; ultraviolet-light; zinc staining
• ISSN: 0864-4551

We recently described a method for recovering nucleic acids separated from polyacrylamide gels, which is based on the sensitive on-gel DNA detection with zinc-imidazole followed by passive elution from 32-mm average-size gel microparticles into the elution buffer. The rationale for the preferential use of DNA micropurified by this method, as compared to DNA obtained by a conventional methodbased on electrophoresis on agarose gels followed by ethidium bromide staining and UV light visualizationwas evaluated. We found that micropurification of two DNA fragments, a 1.2 kbp DNA encoding the kanamycine resistant gene and the pUC19 vector, by both methods produced the same, statistically undistinguishable (P = 0.10)number of colonies when used to transform Escherichia coli XL-1 Blue competent cells. However, the number of transformants obtained in the absence of the insert was significantly lower when the DNA vector was micropurified from the polyacrylamide gel by the zinc-imidazole method (P = 0.0002), which increased the probability of finding desired recombinant colonies in the cloning experiment. These results encouraged the use of our zinc-imidazolebased technique for high quality micropurification of DNA and to overcome situations where DNA purified from conventional LGT agarose (e.g., genes for the class 1 outer membrane proteinboth intact or with a deletion in loop 5and Opc-protein from Neisseria meningitidis 866 strain) failed to give rise to recombinant E. coli cells.