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| Efficient genomic DNA cloning with blue/clear color screening and autosubcloning | | | lBlueSTAR™ is a l replacement vector designed for the production of genomic DNA libraries with efficient cloning of DNA fragments 7–20 kbp in size. The vector contains the unique features of blue/clear visual screening of recombinants and cre-loxP-mediated autosubcloning. Dual polylinker regions contain nine cloning sites that are flanked by T7 and T3 promoters. Novagen offers the uncut vector, as well as prepared arms, ready for high-efficiency cloning of insert DNA with ultra-low nonrecombinant background. Kits are available for cloning with either BamH I or partially filled-in Xho I arms. Another advantage of both lBlueSTAR systems is the protection of inserted genomic DNA from host restriction by the use of the EcoK, mcr- PhageMaker® extracts and host strain ER1647 (1–5).
Blue/clear screening is achieved by the presence of the complete E. coli lacZ gene and control region within the 13 kbp stuffer fragment. The native vector, as well as arms religated with the stuffer fragment, yields phage that produce active b-galactosidase in infected cells. Nonrecombinants are easily identified as blue plaques on plates containing X-gal, whereas phage containing genomic DNA inserts produce clear plaques.
Cloning with Xho I-digested, partially filled arms involves partial fill-in of Sau3A I (or Mbo I or other compatible enzyme) digested genomic DNA, followed by ligation to the vector arms (6). The partial fill-in strategy prevents tandem inserts and religation of arms to the stuffer fragment. Cloning with BamH I-digested (non-filled) arms does not require partial fill-in of genomic DNA prior to ligation, because the BamH I arms can be ligated directly to Sau3A I-digested genomic DNA. Both types of arms have been dephosphorylated to further reduce nonrecombinant background.
Plasmid subclones are easily obtained by plating lBlueSTAR phage on a strain expressing cre recombinase (BM25.8) in the presence of carbenicillin (or ampicillin). These conditions produce colonies that carry the genomic insert in a plasmid excised from the lambda vector. Highly purified Cre Recombinase is also available separately for in vitro manipulations. | | | 1. Wyman, A. R., Wertman, K. F., Barker, D., Helms, C., and Petri, W. H. (1986) Gene 49, 263–271.
2. Wertman, K. F., Wyman, A. R., and Botstein, D. (1986) Gene 49, 253–262.
3. Raleigh, E. A., Trimarchi, R., and Revel, H. (1989) Genetics 122, 279–295.
4. Doherty, J. P., Lindeman, R., Trent, R. J., Graham, M. W., and Woodcock, D. M. (1993) Gene 124, 29–35.
5. Woodcock, D. M., Crowther, P. J., Doherty, J., Jefferson, S., Decruz, E., Noyer-Weidner, M., Smith, S. S., Michael, M. Z., and Graham, M. W. (1989) Nucleic Acids Res. 17, 3469–3478.
6. Zabarovsky, E.R. and Allikmets, R.L. (1986) Gene 42, 119–123. | | | | |
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