The effect of column length, applied voltage, gel type, and concentration on the capillary electrophoresis separation of DNA fragments and polymerase chain reaction products.
This work examines the effect of different parameters on migration time, resolution, and speed of analysis of DNA fragments and PCR products. These parameters include column length, applied voltage, gel type and concentration, and buffer ionic strength. Our results indicate that 1 cm capillary at an applied voltage of 185 V/cm, filled with commercial gel, was adequate for the separation of small DNA fragments in under 1 min. Resolution of large fragments is directly proportional to column length at the same field strength. Also, resolution of large fragments is higher (better) at lower field strength at constant column length. Analysis is fastest (high throughput) using a short capillary and moderate field strength (200 v/cm). CE using a single short capillary (2-7 cm) is comparable to slab gel in throughput, but more economical. The Sigma DNA buffer and hydroxyethyl cellulose liquid gel gave equivalent results in terms of resolution and reproducibility. The Sigma DNA replaceable gel gave reproducible results when used as received or diluted at 60%. In our hands hydroxyethyl cellulose gave more reproducible results than polyacrylamide gel.
Issaq
H J
HJ
SAIC Frederik, NCI-Frederick Cancer Research and Development Center, MD 21702, USA. issaqh@mail.ncifcrf.gov
Chan
K C
KC
Muschik
G M
GM
D002021:Buffers; D004247:DNA / Q000302:isolation & purification*; D004563:Electrochemistry; D019075:Electrophoresis, Capillary / Q000379:methods*; D005782:Gels / Q000737:chemistry; D010316:Particle Size; D016133:Polymerase Chain Reaction
D002021:Buffers; D005782:Gels; D004247:DNA