Zero Blunt® TOPO® PCR Cloning kits for subcloning provide a highly efficient, 5-minute cloning reaction for the direct insertion of blunt-ended PCR products amplified with proofreading thermostable polymerases into a plasmid vector. Each kit has the Zero Blunt® TOPO® vector containing the ccdB gene for positive selection, only permitting growth of plasmid vectors with recombinants. Zero® Blunt® TOPO® kits are available with a variety of competent cells, or without competent cells, depending on your needs and budget.
• Fast and easy—go from PCR to clones in just 3 steps, including a 5-minute cloning reaction
• Efficient—obtain up to 95% clones with correct insert
• Proven—enabling reliable performance for over a decade with over 20,000 citations
• Simple—no ligase, post-PCR procedures, or PCR primers containing specific sequences required
• Accurate—for use with high fidelity DNA polymerases, no need for A-overhang on PCR product
pCR™Blunt II-TOPO® Vector—Designed for Proofreading Polymerases
The pCR™Blunt II-TOPO® vector is designed for the cloning of blunt-ended PCR products generated by thermostable proofreading polymerases such as Platinum® Pfx DNA Polymerase. The pCR™Blunt 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
• ccdB gene, allowing for direct selection of recombinants
pCR™Blunt II-TOPO® Clone Selection
pCR™Blunt 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.
The Most Widely Used Cloning Kit
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.
Kit Options: Zero Blunt® TOPO® PCR Cloning® Kit for Subcloning
Zero Blunt® TOPO® PCR Cloning kits 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. Nos. K2800-J10, K2800-20, K2800-40)
• High-efficiency cloning: TOP10 Electrocomp™ cells (Cat. Nos. 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 cells (Cat. Nos. 451254, 450245)
We also offer 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.
|450245||Unit Size: 25 reactions|
|451245||Unit Size: 10 reactions|