Reversible gels for electrophoresis and isolation of DNA.
Here, the application of the gel-forming carbohydrate polymer, gellan gum, for the electrophoresis and isolation of DNA is detailed. Gellan gun forms gels in the presence of divalent metal cations, and the gels can be converted back to a solution by the addition of a chelating agent such as EDTA. Also, gellan electrophoresis gels can be formed using diamines. These gels are reversible by increasing the pH, which results in the deprotonation of the diamine. Gellan electrophoresis gels were used for separations at concentrations as low as 0.03%. Native gellan electrophoresis gels have significant electroosmosis and were generally run overnight. A gellan electrophoresis gel (0.1%) showed good resolution of DNA from approximately 50-1 kbp. The addition of linear polymers, such as hydroxethyl cellulose, to the gellan gum before casting greatly reduced the electroosmosis. The additional polymer increased the resolution of low-molecular-weight DNA down to approximately 200 bp and allowed gels to be run in a few hours. DNA isolated from gellan electrophoresis gels could be cut by common restriction enzymes and ligated in the presence of the gellan gum. The presence of gellan gum did not significantly inhibit the transformation of competent E. coli cells by plasmid DNA.
Cole
K D
KD
National Institute of Standards and Technology, Gaithersburg, MD, USA.
D001709:Biotechnology; D002413:Cations, Divalent; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification*; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D003959:Diamines; D004586:Electrophoresis / Q000379:methods*; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics; D005069:Evaluation Studies as Topic; D005782:Gels; D008970:Molecular Weight; D011135:Polysaccharides, Bacterial; D014170:Transformation, Genetic
D002413:Cations, Divalent; D004269:DNA, Bacterial; D003959:Diamines; D005782:Gels; D011135:Polysaccharides, Bacterial; C048288:gellan gum; D004247:DNA; D004262:DNA Restriction Enzymes