70635 Introductory pETBlue-2 Perfectly Blunt® Cloning Kit - Novagen

70635
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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, also consider the pSTBlue-1 Perfectly Blunt Giga Cloning Kit (Cat. No. 71229).

      The pETBlue™-2 vector is designed to identify recombinants by traditional blue/white screening while also allowing T7lac promoter based expression of target genes. Screening is independent of expression because the T7lac expression promoter is in an opposed orientation relative to the E. coli promoter that mediates blue/white screening. pETBlue-2 defines the open reading frame and inserts must be cloned in-frame if expression is desired. The vector features an expanded multiple cloning site (MCS) and optional C-terminal HSV•Tag® and His•Tag® sequences. The sequence is numbered from the first base of the T7 promoter sequence.


      In addition to the pETBlue™ Systems, which contain uncut plasmids, pETBlue vectors are available in formats ready for insertion of PCR products. Two types of kits are available:
      AccepTor™ Vector Kits and Perfectly Blunt® Cloning Kits. The AccepTor Vector Kit enables direct insertion of DNA containing single 3′-dA overhangs, such as those amplified with non-proofreading DNA polymerases (e.g., NovaTaq™ DNA Polymerase), while the Perfectly Blunt Cloning Kits are designed for cloning inserts having any type of DNA end.

      Catalogue Number70635
      Brand Family Novagen®
      Features and benefits
      • No restriction enzymes or special primers
      • Compatible with any DNA polymerase
      • Independent of 3ʹ-dA addition or sequence context of DNA ends
      • Blue/white screening with seven different vectors, including pETBlue expression vectors
      • Simple protocol takes less than one hour* from PCR product to plating transformants

      * All times listed refer to kits using NovaBlue Singles™ Competent Cells and ampicillin or chloramphenicol selection. Giga kits, which use NovaBlue GigaSingles™ Competent Cells, require one hour for outgrowth.

      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.

      Product Information
      0.5 µgBlunt Vector
      10 µlPositive Control Insert
      100 µlEnd Conversion Mix
      100 UT4 DNA Ligase
      1.5 mlNuclease-free Water
      11 × 50 µlNovaBlue Singles Competent Cells
      3 × 2 mlSOC Medium
      10 µlTest Plasmid
      0.2 mlTuner™(DE3)pLacI Competent Cells
      Quality LevelMQ100
      Applications
      Biological Information
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Storage and Shipping Information
      Ship Code Dry Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Storage ≤ -70°C
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Specifications

      Documentation

      Introductory pETBlue-2 Perfectly Blunt® Cloning Kit - Novagen Certificates of Analysis

      TitleLot Number
      70635

      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
    • Michelle B. Ryndak, et al. (2005) Role of predicted transmembrane domains for type III translocation, pore formation, and signaling by theYersinia pseudotuberculosis YopB protein. Infection and Immunity 73, 2433-2443.
    • Yue Zhang, et al. (2005) Inhibition of MAPK and NF-κB pathways is necessary for rapid apoptosis in macrophages infected with Yersinia. Journal of Immunology 174, 7939-7949.
    • Yasuyo Yamazaki, et al. (2003) Camptothecin biosynthetic genes in hairy roots of Ophiorrhiza pumila: cloning, characterization and differential expression in tissues and by stress compounds. Plant and Cell Physiology 44, 395-403.
    • User Protocols

      Title
      TB183 Perfectly Blunt® Cloning Kits

      Vector Map

      Title
      TB259VM pETBlue™-2 Vector Map

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      Categories

      Life Science Research > Genomic Analysis > Transfection and Protein Expression > Bacterial Expression > Bacterial Expression Vectors
      Life Science Research > Genomic Analysis > DNA Preparation & Cloning > Cloning > Cloning Kits