Preparation and purification of restriction endonuclease fragments

Dilute the DNA extract obtained by the crush and soak method with 4 vols. H20 and pass through a small (1 ml) column of DEAE-cellulose, DE-52, equilibrated with five-fold diluted gel-extraction buffer. Wash the column with a further 2 ml buffer and elute the absorbed DNA with 1 ml of 1.1 M Nad in diluted extraction buffer and precipitate by the addition of 3 vols. ethanol. The recovery of DNA is 95% and is free from all traces of gel and acrylamide monomer. DNA extracted by electroelution may be purified in the same manner except that prior dilution is not necessary and the column is equilibrated with the electroelution buffer. 

Usually DNA precipitated by the addition of ethanol is recovered by centrifuging in an Eppendorf centrifuge (Model 5412) at 

000 rpm for 10 min. Where very small quantities of DNA have to be recovered, centrifugation at 45 K rpm for 1 h at 4°C in a SW-60 rotor (or equivalent) is recommended. As much supernatant as possible is drained from the pellet (often invisible) which is finally washed once with 95% ethanol and recentrifuged for the same speed and time. Care is needed to avoid dislodging the pellet after the ethanol wash. After drying, the DNA is dissolved in a small volume of water and stored frozen at —20°C. 

Extraction of the DNA may be achieved by a variety of methods (see e.g. Smith, 1980). Two procedures which are rapid and convenient are described below  

Method 1 takes advantage of the properties of a low melting temperature agarose (Bethesda Research Laboratories, Rockville, Md., USA). The gel slice is melted at 70°C and an equal volume of 10 mM Tris-HCl, (pH 7.6), 0.1 mM-EDTA added and the solution extracted with the same volume of water-saturated phenol in one or more 1.5 ml Eppendorf centrifuge tubes. After brief centrifugation the aqueous layer is removed and the phenol extraction repeated a further 2-3 times. Finally the aqueous phase is extrac- 

---