An apparatus for electrophoretic capture and recovery of circular DNA in thin layers.
An apparatus was designed for the electrophoretic capture and recovery of circular DNA in thin layers (membranes). Rapid separations were done by the use of a low-conductivity buffer and high electric field strengths. Two methods that specifically retain circular DNA in the membranes were demonstrated using the supercoiled and open circular forms of two plasmids with sizes of 4.4 and 13 kbp. Electrophoretic trapping (by an impalement mechanism) in agarose-gel-filled membranes used electric field strength to immobilize circular DNA in the membranes. The other method of capture utilized the greatly reduced electrophoretic mobility of circular DNA in membranes composed of agarose and the linear polymer hydroxyethylcellulose. The reduction in electrophoretic mobility was not dependent upon the electric field strength, distinguishing it from electrophoretic trapping. Trapping of circular DNA in the membranes followed by size analysis using agarose-gel electrophoresis could be used as a two-dimensional separation tool for the analysis of complex mixtures. Captured DNA was recovered by two methods: (i). centrifugation of membranes made with low-melting-point agarose resulted in a gel slurry that could be heated to release the DNA; (ii). electroelution of the membranes. Electroelution was done by using an electrode that was isolated from the DNA by a 'barrier' membrane. A non-trapping electric field was used to reverse the DNA out of the membrane into a small volume of buffer above the membrane.
Cole
Kenneth D
KD
Bioprocess Engineering Group, Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA. Kenneth.Cole@nist.gov
D000327:Adsorption; D002498:Centrifugation / Q000379:methods*; D004270:DNA, Circular / Q000737:chemistry* / Q000302:isolation & purification*; D004563:Electrochemistry / Q000295:instrumentation / Q000379:methods; D004587:Electrophoresis, Agar Gel / Q000295:instrumentation* / Q000379:methods*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D008567:Membranes, Artificial
D004270:DNA, Circular; D008567:Membranes, Artificial