73] Fractionation of DNA on methylated albumin column

Abstract

This chapter describes the principle, reagents, and procedure involved in the fractionation of DNA on methylated albumin column. Methylated albumin kieselguhr (MAK) column fractionates DNA by its size, content of hydrogen bonding, and base composition. The size of the column can be variable depending on the amount of DNA to be fractionated. Maximum capacity of the column with good fractionation is 1 mg of DNA per 10 ml of the MAK suspension. The packed column is approximately half the MAK suspension. The height of the MAK packed in the column should be between 1 cm and maximum 5 cm. The chapter describes two ways of elution: stepwise and gradient. For preparatory purposes, the stepwise elution is most useful, whereas for analytical purposes the gradient elution is usually more informative. To obtain concentrated DNA fractions, it is advisable to work with an amount of DNA close to the maximum capacity of the column (1 mg of DNA per 10 ml of MAK suspension). Recovery of native DNA is usually 100%, but that of denatured DNA is 50-70%. Native DNA elutes between 0.6M and 0.75M NaCl, whereas denatured DNA elutes between 0.8 M and 0.95 M NaCl, in the molecular weight range of 10 X 10 6 to 20 X 10 6 .