70183 Introductory pT7Blue Perfectly Blunt® Cloning Kit - Novagen

70183
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      Description
      Overview

      The Perfectly Blunt® Cloning Kits are designed for simplified cloning of DNA generated by PCR using any type of DNA polymerase. This approach enables the use of high-fidelity proofreading enzymes for amplification, thus decreasing the probability of generating mutations in the target sequence. In addition, under many conditions blunt cloning is more efficient than T-cloning, most likely due to the observation that the efficiency of single dA addition by Taq DNA polymerase varies significantly depending on the sequence context of the DNA ends, and even the number of PCR cycles performed (Novy 1996, Clark 1998, Brownstein 1996, Magnuson, 1996, Hu 1993).

      With the Perfectly Blunt cloning protocol, you can go from PCR product to plating transformants in less than one hour with minimal hands-on time. The finished PCR product is converted to a blunt, phosphorylated form in a 15-minute reaction using premixed reagents. Following a 5-minute heat inactivation step, the treated insert is combined with the ready-to-use vector and ligated in an optimized 15-minute reaction. An exclusive 8-minute transformation procedure using highly efficient NovaBlue Singles™ Competent Cells (Cat. No. 70181) generates recombinant colonies that are easily visualized by blue/white screening.

      Note that the Perfectly Blunt method is not limited to cloning PCR products; these kits are also suitable for cloning restriction fragments, cDNA, or sheared DNA with the same protocols.

      Seven different vectors are available in Perfectly Blunt® Cloning Kits. Vector choices include those designed for general cloning, sequencing, optimal in vitro transcription/translation, and optimal protein expression in E. coli. Each vector is available in a kit containing sufficient reagents for 10, 20, or 40 reactions.

      “Vector only” kits are also available in 20- and 40-reaction sizes without ligase and competent cells. For higher-efficiency competent cells, see also pSTBlue-1 Perfectly Blunt Giga Cloning Kit 71229).

      The pT7Blue vector features the pUC19 backbone (including high-copy number origin of replication and lac sequences), a T7 promoter, f1 origin of replication, and modified multiple cloning region. The multiple cloning region contains an EcoR V site used for T-cloning flanked by an Nde I site, which allows inserts to be conveniently subcloned into the Nde I site of many pET vectors.



      Catalogue Number70183
      Brand Family Novagen®
      References
      References

      Novy, R.E., et al. 1996. inNovations 6, 7. Clark, J.M. 1988. Nucleic Acids Res. 16, 9677. Brownstein, J.M., et al. 1996. BioTechniques 20, 1004. Magnuson, V.L., et al. 1996. BioTechniques 21, 700. Hu, G. 1993. DNA and Cell Biology 12, 763.

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      Quality LevelMQ100
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      Ship Code Dry Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Storage ≤ -70°C
      Avoid freeze/thaw Avoid freeze/thaw
      Do not freeze Ok to freeze
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      Introductory pT7Blue Perfectly Blunt® Cloning Kit - Novagen Certificates of Analysis

      TitleLot Number
      70183

      References

      Reference overview

      Novy, R.E., et al. 1996. inNovations 6, 7. Clark, J.M. 1988. Nucleic Acids Res. 16, 9677. Brownstein, J.M., et al. 1996. BioTechniques 20, 1004. Magnuson, V.L., et al. 1996. BioTechniques 21, 700. Hu, G. 1993. DNA and Cell Biology 12, 763.

      Citations

      Title
    • Ekaterina Bogdanova, et al. (2008) Transcription regulation of the type II restriction-modification system AhdI. Nucleic Acids Research 36, 1429-1442.
    • Miho Hirabayashi, et al. (2005) Transformation of skeletal muscle from fast- to slow-twitch during acquisition of cold tolerance in the chick. Endocrinology 146, 399-405.
    • J. K. Jang, T. Rahman and K. S. McKim. (2005) The kinesinlike protein subito contributes to central spindle assembly and organization of the meiotic spindle in Drosophila oocytes. 16, 4684-4694.
    • Rahul Pal, et al. (2005) Evidence for multiple shared antigenic determinants within Ro60 and other lupus-related ribonucleoprotein autoantigens in human autoimmune responses. Journal of Immunology 175, 7669-7677.
    • User Protocols

      Title
      TB183 Perfectly Blunt® Cloning Kits

      Vector Map

      Title
      TB017VM pT7Blue Vector Map

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      Categories

      Life Science Research > Genomic Analysis > DNA Preparation & Cloning > Cloning > Cloning Kits