Zero Blunt® TOPO® PCR Cloning provides a highly efficient, 5 minute, one-step cloning strategy ("TOPO® Cloning") for the direct insertion of blunt-end 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 and are available with a variety of competent cells, or no competent cells, depending on your needs and budget.
Fast and Easy—Go from PCR-to-clones in just 3 steps and in as little as 5 minutes hands-on time
Efficient—Achieve up to 95% clones with correct insert
Proven—Reliable performance for over a decade with over 4000 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
— pCR®-Blunt II-TOPO® contains the ccdB gene allowing for direct selection of recombinants.
CLONING METHOD: TOPO® Cloning
— Topoisomerase I based 5 minute ligation of blunt-end 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 — Efficiency with proofreading polymerase
The pCR®Blunt II-TOPO® vector is designed to clone blunt-end PCR products generated bythermostable 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 (Bernard et al., 1994). 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.
With up to 95% of clones carrying the desired insert, you can screen less clones to help you save time and money. The pCR®Blunt II-TOPO® vector used in this kit comes with lethal ccdB gene for efficient selection of blunt-end 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-end PCR products into a plasmid vector can be purchased with a variety of competent cells that deliver different advantages depending on what your needs are:
General cloning: TOP10 (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.
TOPO® products are For Research Use Only. Not intended for any animal or human therapeutic of diagnostic use.