Zero Blunt® TOPO® PCR Cloning Kit provides a highly efficient, 5-minute, one-step cloning strategy ("TOPO® Cloning") 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 (see figure) 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. Features of the Zero® Blunt® TOPO® PCR Cloning Kits:
Fast and easy—go from PCR to clones in just 3 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.
Zero Blunt® TOPO® Cloning Kits—overview
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, or use your own.
pCR™Blunt II-TOPO® vector—designed for proofreading polymerases The pCR™Blunt II-TOPO® vector is designed to clone 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
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.
Efficient cloning With up to 95% of clones carrying the desired insert, you can screen less clones, saving you time and money. The pCR™Blunt II-TOPO® vector used in this kit comes with lethal ccdB gene for efficient selection of blunt-ended PCR recombinants products generated bythermostable 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. No. 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)
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.
For Research Use Only. Not for use in diagnostic procedures.
Antibiotic Resistance Bacterial:
Kanamycin (KanR), Zeocin™ (ZeoR)
Bacterial or Yeast Strain:
TOPO Cloning Vectors
Contents & storage
Box 1: • Linearized and topoisomerase I-activated pCR™Blunt II-TOPO® vector • Salt solution • dNTPs • Control template • M13 forward and reverse primers • Sterile water
Store at -5 to -30°C. All reagents are stable for 6 months when properly stored.
Box 2: • One Shot® Chemically Competent or Electrocomp™ E. coli • S.O.C. medium • Supercoiled pUC19 control plasmid