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Several methods have been reported for the small scale preparation of plasmid DNA for sequencing (1-9), but little attention has been given to the effect of the E. coli host strain on the quality of the sequence obtained from double-stranded DNA. It has been observed that endonuclease A (endA) E.coli strains may produce nicked plasmid DNA resulting in 'shadow' bands on sequencing gels (1, 8), but no systematic evaluation of this phenomenon has been performed. We have found that of ten host strains tested, DH5a, JM109 and SURE cells consistently produced the highest yields of good quality plasmid DNA for double-stranded sequencing, whereas two of four endA strains tested (NM522 and TG-1) exhibited significantly increased background on sequencing gels. Plasmid DNA was prepared from E.coli host strains DH5a and DH5aF' (Bethesda Research Laboratories), HB101 (10), JM101 (11), JM105 (11), JM109 (11), NM522 (12), SURE (Stratagene), TGI (Amersham), and XL 1-Blue (Stratagene) transformed with pBluescriptll KS(+) (Stratagene). DNA was isolated from 1.5 ml of the overnight cultures by the alkaline lysis mini-preparation method of Sambrook et al. (6) with and without the optional phenolxhloroform.isoamyl alcohol (24:24:1) extraction. Dideoxy sequencing reactions were performed on alkali-denatured plasmid with 10 ng of either T7 or T3 oligonucleotide primer (Stratagene) and Sequenase v.2 (US Biochemical) as described in the Sequenase protocol. Gel electrophoresis of sequencing reactions was performed as described previously (13). All plasmids were isolated and sequenced three times with similar results (data submitted but not shown). We initiated these studies to determine which commonly used E. coli host strains produce the highest yields of plasmid DNA for good quality sequencing. DNA from all strains prepared with

E.colihost strains significantly affect the quality of small scale plasmid DNA preparations used for sequencing