Improved manufacture and application of an agarose magnetizable solid-phase support.
A simple, semiautomated, nonhazardous procedure for the production of a magnetizable solid-phase support (MSPS) has been developed based on the extrusion of molten agarose-iron oxide mixtures, which enables manufacture of a range of differently sized spherical agarose-iron oxide beads. This system has enabled scale-up of an original manufacture procedure and reproducible preparation of kg quantities of MSPS suitable for biomolecular purifications. An improved protocol for the isolation of plasmid DNA directly from cell lysates using this MSPS, derivatized with diethylaminoethyl (DEAE) groups, is reported. This involves a modified alkaline lysis, followed by adsorption to and elution from the support, yielding plasmid DNA of a purity comparable with, or better than, other methods of plasmid isolation. Using the same procedure, plasmid DNA can be isolated from bacterial cell culture volumes of 1.5 mL and 100 mL with equal efficiency and purity.
Davies
M J
MJ
Dept. of Chemical and Life Sciences, University of Greenwich, London.
Smethurst
D E
DE
Howard
K M
KM
Todd
M
M
Higgins
L M
LM
Bruce
I J
IJ
D002850:Chromatography, Gel / Q000379:methods*; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics; D005290:Ferric Compounds; D008280:Magnetics; D008863:Microspheres; D010957:Plasmids / Q000235:genetics; D012685:Sepharose
D004269:DNA, Bacterial; D005290:Ferric Compounds; C000499:ferric oxide; D012685:Sepharose