Electrophoretic separation of DNA using a new matrix in uncoated capillaries.
A new separation matrix, consisting of polymer poly(N-isopropylacrylamide) (PNIPAM) and small molecule additive mannitol, was used for double-stranded (ds) DNA and plasmid DNA separation by capillary electrophoresis. The matrix had a low viscosity, which made it very easy to handle. The additive mannitol dramatically enhanced the sieving performance of PNIPAM in TBE buffer. The optimal mannitol concentration 6% in polymer solution, was determined with the consideration of both speed and resolution. A resolution of 0.95 was achieved on the separation of 271/281 bp in the phiX174/HaeIII digest by using 1.5% PNIPAM + 6% mannitol, while the supercoiled, linear and nicked conformers of lambda plasmid were separated in 1% PNIPAM + 6% mannitol, demonstrating the potential use of this new matrix for effective DNA separations. The dramatic impact of mannitol on sieving performance of PNIPAM solution was investigated. pH dependent self-coating ability of PNIPAM was revealed. The presence of mannitol in TBE buffer decreased the pH of the buffer, which led to more efficient self-coating ability of PNIPAM probable due to the formation of hydrogen bonds between PNIPAM molecules and silanol groups at the silica wall.
Zhou
Ping
P
College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China. zbping@whu.edu.cn
Yu
Shengbing
S
Liu
Zhonghua
Z
Hu
Jiming
J
Deng
Yanzhuo
Y
D000180:Acrylic Resins; D004247:DNA / Q000302:isolation & purification*; D019075:Electrophoresis, Capillary / Q000295:instrumentation* / Q000379:methods*; D008353:Mannitol; D010957:Plasmids / Q000235:genetics; D014783:Viscosity
D000180:Acrylic Resins; C052970:poly-N-isopropylacrylamide; D008353:Mannitol; D004247:DNA