OPTIMIZATION OF CAPILLARY ELECTROPHORETIC SEPARATION OF DNA FRAGMENTS BASED ON POLYMER FILLED CAPILLARIES

The separation of Phi X174RF DNA-Hae III fragments is studied in a 0.5% methycellulose polymer solution as a function of temperature and applied voltage. The migration times decreased with both increasing temperature and increasing field strength, as expected. However, the relative migration rates of the fragments (selectivity) did not change with temperature but are affected by the applied field. A clear transition from the Ogston mechanism to the reptation mechanism is observed. The use of 0.5% methyl cellulose and 202 mM berate buffer at pH 8.2 allows the separation of the 271/281 base pair fragments, even without the addition in intercalating agents. At 700 V/cm and 20 degrees C, complete separation of all fragments is achieved in less than 4 min with an average plate number of 2.5 . 10(6)/m.