Zero Blunt™ TOPO™ PCR Cloning kits for sequencing provide a highly efficient, 5-minute cloning reaction for the direct insertion of proofreading polymerase-amplified, blunt-ended PCR products into a plasmid vector for sequencing. Each kit uses the pCR™4Blunt-TOPO™ vector with specially designed sequencing primer sites that return more insert sequence and less vector sequence from each reaction. These kits include the tools necessary to clone and select recombinant vectors containing your PCR fragment of choice.
• 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 are required
• Accurate—for use with high fidelity DNA polymerases, no need for A-overhang on PCR product
pCR™4Blunt-TOPO™ Vector—Optimized for Sequencing
The pCR™4Blunt-TOPO™ vector has a minimized multiple cloning site and, thus, a shorter distance between the sequencing primer sites and the insert site (as little as 33 bp). So sequencing reactions produce less vector sequence and more insert sequence. The pCR™4Blunt TOPO™ vector has sites for 4 common sequencing primers: M13 forward, M13 reverse, T7, and T3. The kits include an aliquot of each.
pCR™4Blunt-TOPO™ Clone Selection and Manipulation
The pCR™4Blunt-TOPO™ vector contains both ampicillin and kanamycin resistance markers and a LacZα-ccdB gene fusion for positive selection. The vector’s minimized multiple cloning site still includes flanking EcoRI sites for simplified excision of cloned PCR products and a unique Sse8387I site for generation of nested deletions prior to sequencing. T7 and T3 promoters are also present for in vitro transcription.
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 fewer clones, which helps save time and money. The pCR™4Blunt-TOPO™ vector has no overhangs for efficient ligation of PCR products created by proofreading thermostable polymerases that leave blunt-ended PCR products.
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 Sequencing
Zero Blunt™ TOPO™ PCR Cloning kits for sequencing can be purchased with a variety of competent cells that deliver different advantages depending upon your needs:
• General cloning: TOP10 cells (Cat. Nos. K2875-J10, K2875-20, K2875-40)
• High-efficiency cloning: TOP10 Electrocomp™ cells (Cat. Nos. K2880-20, K2880-40)
• General cloning, bacteriophage T1 resistance: DH5α-T1R (Cat. No. K2895-20)
• Fast growth: Mach1™-T1R chemically competent E. coli (Cat. No. K2835-20)• Provide your own cells (Cat. Nos. 450031, 450159)
For Research Use Only. Not for use in diagnostic procedures.