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Comparison of two gel electrophoresis-based DNA micropurification methods for molecular cloning

Abstract:

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-μm 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 method - based on electrophoresis on agarose gels followed by ethidium bromide staining and UV light visualization - was 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-imidazole-based 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 protein - both intact or with a deletion in loop 5 - and Opc-protein from Neisseria meningitidis 866 strain) failed to give rise to recombinant E. coil cells.