Includes: Linearized and topoisomerase I-activated pCR™Blunt II-TOPO™ vector; Salt solution; dNTPs; Control template; M13 forward and reverse primers; Sterile water
Includes the Zero Blunt TOPO vector containing the ccdB gene for positive selection, only permitting growth of plasmid vectors with recombinants. Resistant to Kanamycin (KanR) and Zeocin™ (ZeoR). pCRBlunt II-TOPO vector: designed for proofreading polymerases The pCRBlunt II-TOPO vector is designed to clone blunt-ended PCR products generated by thermostable proofreading polymerases such as Platinum™ Pfx DNA Polymerase. The pCRBlunt II-TOPO vector contains:
EcoRI sites flanking the PCR product insertion site for easy excision of inserts
Kanamycin and Zeocin resistance genes for your choice of selection in E. coli
SP6 promoter/primer site for in vitro RNA transcription and sequencing
M13 forward and reverse primer sites for sequencing or PCR screening
pCRBlunt II-TOPO clone selection pCRBlunt II-TOPO allows for direct selection of recombinants via disruption of the lethal E. coli gene, ccdB. The vector contains the ccdB gene fused to the C-terminus of the LacZα fragment. Ligation of a blunt-end PCR product disrupts expression of the LacZα-ccdB gene fusion, permitting growth of only positive recombinants upon transformation. Cells that contain non-recombinant vector are killed upon plating. Therefore, blue/white screening is not required. Simplified TOPO-based cloning Using TOPO cloning technology, there is no need for PCR primers containing specific sequences, post-PCR procedures, vector preparation, or other time-intensive DNA manipulation steps. Just add your PCR reaction straight to the provided topoisomerase-charged vector, incubate 5 minutes, and transform E. coli competent cells. Efficient cloning With up to 95% of clones carrying the desired insert, you can screen fewer clones, saving you time and money. The pCRBlunt II-TOPO vector used in this kit comes with lethal ccdB gene for efficient selection of blunt-ended PCR recombinants products generated by thermostable proofreading polymerases. The standard in cloning When it comes to cloning, TOPO™ cloning technology has been a reliable partner for thousands of scientists for over ten years. Fast, simple-to-use, and efficient, TOPO™ cloning has been applied to many different vectors for a wide array of applications. Zero Blunt TOPO PCR Cloning Kit options The Zero Blunt TOPO PCR Cloning Kit for direct insertion of blunt-ended PCR products into a plasmid vector can be purchased with a variety of competent cells that deliver different advantages depending upon your needs General cloning: TOP10 cells (Cat. No. K2800-20, K2800-40) High-efficiency cloning: TOP10 Electrocomp™ Cells (Cat. No. K2860-01, K2860-40) General cloning, bacteriophage T1 resistance: DH5α-T1R (Cat. Nos. K2820-20, K2820-40) Fast growth: Mach1™-T1R Chemically Competent E. Coli (Cat. No. K2830-20) Provide your own: for flexibility and to save money (Cat. No. 450254) A version of the kit that includes a PureLink™ Quick Plasmid Miniprep Kit (Cat. No. K2800-02) for use in isolation of clean, sequencing-ready, recombinant plasmid is also available.
Fast and easy: go from PCR to clones in just three steps and in as little as 5 minutes hands-on time
Efficient: obtain up to 95% clones with correct insert
Proven: reliable performance for over a decade with over 4,000 citations
Simple: no ligase, post-PCR procedures, or PCR primers containing specific sequences are required
Vector: pCR™Blunt II-TOPO vector: contains the ccdB gene, allowing for direct selection of recombinants
Cloning method: TOPO cloning: Topoisomerase I–based, 5-minute ligation of blunt-ended PCR products amplified with proofreading thermostable polymerases
Competent cells: Various options: choose from kits with either general, high-efficiency, bacteriophage T1-resistant, fast-growing competent cells (available separately) or use your own
