Purification of cDNA for cloning using high performance gel permeation chromatography.

The use of HPLC in the separation of DNA fragments is well established (1). We have adapted these techniques to the separation of cDNA from linker sequences present during lambda cloning. Short linker sequences containing an EcoRl restriction site are coupled to blunt ended cDNA, excess linkers are cleaved using EcoRl. The efficiency of cloning into the lambda EcoRl site is improved by the removal of competing linker fragments, usually achieved by affinity chromatography or gel electrophoresis. We have used gel-permeation HPLC to improve the purity of the cDNA obtained during this separation step. This method also allows the exclusion of short DNA sequences from the ligation reaction, thus increasing the average molecular weight of the inserts cloned. EcoRl linkers were added to the cDNA according to the method of Kurtz (2). The restricted cDNA/linker mixture was precipitated and the pellet resuspended in lOOjil of TE buffer (lOmM Tris. lmM EDTA pH 7.0); this sample was loaded onto an Ultropac TSK G4000-SW (7.5x600 mm) column and eluted with TE buffer at 0.1 ml/min. The column effluent was monitored at 254 nm and 0.5 ml fractions collected. A typical elution profile is shown in fig 1. cDNA in the void volume peak was precipitated using sodium acetate, ethanol and glycogen (3). After resuspension into TE buffer, cDNA was cloned into x gtlO and Xgtll. Cloning into A gtlO gave 3 xl0-> plaques/ng cDNA, 51% of which were recombinant. The size of the inserts ranged from 1.7-3.3 Kbp. Cloning into Xgtll gave 4.5 xlO^ plaques/ug cDNA, 77% of which were recombinant. The size of the inserts ranged from 0.1-1.3 Kbp. This method has shown