Enhanced capacity for electrophoretic capture of plasmid DNA by agarase treatment of agarose gels.
Agarase was used investigate the effect of increasing the number of polymer ends on the electrophoretic trapping of circular DNA in agarose gels. The electric field strength required to trap circular DNA was found to be the same in control and treated gels, indicating the treatment did not result in longer traps. Loading experiments indicated that treated gels had a significantly higher capacity for the open circular DNA. Electrophoretic mobility measurements using pulsed fields indicated a higher density of active traps for treated gels compared to controls. Linear dichroism experiments showed that impalement occurred by a fast and a slow process that had characteristic time constants in the one and tens of seconds ranges, respectively. The open circular DNA was more efficiently impaled in the treated gel compared to the control. The considerably higher efficiency of trapping indicated that agarase treatment increased the concentration of traps substantially.
Cole
K D
KD
Bioprocess Engineering Group, Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, USA.
Akerman
B
B
D004247:DNA / Q000302:isolation & purification*; D004270:DNA, Circular / Q000737:chemistry / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D006026:Glycoside Hydrolases / Q000737:chemistry*; D008970:Molecular Weight; D010957:Plasmids / Q000737:chemistry*; D016062:Porosity; D012685:Sepharose / Q000737:chemistry*
D004270:DNA, Circular; D004247:DNA; D012685:Sepharose; D006026:Glycoside Hydrolases; C019624:agarase