A semi-automated method for preparing high-quality plasmid templates in 96-well format ready for automated DNA sequencing

Abstract

Summary This method has been successfully used to prepare plas-mid templates from conventional Escherichia coli strains[INV alpha, TOP-10 (Invitrogen), XL1-Blue (Stratagene),and HB101 (Life Technologies GIBCO-BRL)] and by us-ing many different recombinant plasmid vectors (pUC se-ries, pBlueScriptSK−, TA vector and pGEM). We did notobserve significant lane-to-lane variation in DNA yield.The best DNA yield was obtained by processing 70 µ lofbacteria corresponding to 700 µ l LB growth. In compari-son with the previously mentioned method (Ref. 4), con-sistent good-quality sequencing reactions were obtainedonly if DNA elution was performed twice in TE-RNase so-lution (Fig. 2). In general, we use one-third of the en-tire preparation, corresponding to 300 ng/template, for adye primer cycle sequencing reaction, using ET primers(Amersham) and Thermo sequenase enzyme (Amersham)(Ref. 5; Fig. 3). The time spent per run was estimatedat 1 h per 96 plasmid templates. The price for eachindividual DNA preparation has been estimated to be0.50 US dollars and includes Beckman tips, solutionsand the fiberglass multiscreen plate. This is considerablycheaper than most kits currently available. The 96 double-strand Biomek 2000 plasmid preparation method is avail-able by downloading the zip file from our Web site:http://www.tigem.it/TIGEM/SEQCORE/protocols.html