5-minute cloning with positive selection

TOPO® TA Cloning kits for sequencing utlize our popular pCR™4-TOPO® TA Vector for the direct ligation of Taq-amplified PCR products in a 5-minute, benchtop reaction yielding up to 95% recombinants.

pCR™4-TOPO® allows you to directly select recombinants by disrupting the lethal E. coli gene, ccdB. The vector contains the ccdB gene fused to the C-terminus of the LacZα fragment. Ligating a PCR product disrupts expression of the lacZα-ccdB gene fusion permitting growth of only positive recombinants upon transformation in competent cells that contain non-recombinant vector are killed upon plating. Therefore, blue/white screening is not required.

立即订购

 The pCR™4-TOPO® TA Vector—Optimized for Sequencing

  • Minimal multiple cloning site to shorten the distance between sequencing primer sites and the insert site to as little as 33 bp
  • Both ampicillin and kanamycin resistance markers and a LacZα-ccdB gene fusion for positive selection and blue⁄white screening
  • 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.
 

The pCR™4-TOPO® TA Vector

The pCR™4-TOPO® TA Vector

Performance and value

Each TOPO® TA Cloning® Kit for sequencing contains linearized and topoisomerase I-activated pCR™4-TOPO® TA Vector, Salt Solution, dNTPs, Control Template and Primers, M13 Forward and Reverse Primers, One Shot® Chemically Competent or Electrocomp™ E. coli, S.O.C. Medium, and a supercoiled control plasmid. The TOPO® TA Cloning® Kit for sequencing without competent cells allows the flexibility of using your own strain for increased flexibility so competent cells and S.O.C. Medium are not provided.

In addition, the TOPO® TA Cloning® Kit for Sequencing, with One Shot® TOP10 Chemically Competent E. coli, and PureLink™ Quick Plasmid Miniprep Kit allows for for fast plasmid purification of your TOPO®-cloned inserts for downstream analysis.

Using a proofreading enzyme?

Thermostable polymerases containing extensive 3´ to 5´ exonuclease activity, such as Platinum® Pfx, do not leave 3´ A-overhangs. PCR products generated with Taq polymerase have a high efficiency of cloning in the TOPO® TA subcloning kits because the 3´ A-overhangs are not removed. However, if you use a proofreading polymerase or wish to clone blunt-ended fragments, you can add 3´ A-overhangs by incubating with Taq at the end of your cycling program.

Alternatively, you may want to try Zero Blunt® TOPO® PCR Cloning Kits. These kits offer fast and efficient cloning of blunt-end PCR products generated using thermostable, proofreading polymerases. They also utilize the ccdB gene for positive selection.

订购信息

目录号 产品描述 Competent cells supplied 反应次数  
K4575J10
TOPO® TA Cloning® Kit for Sequencing, with One Shot® TOP10 Chemically Competent E. coli
TOP10 (genotype) 10 立即订购
450030
TOPO® TA Cloning® Kit for Sequencing, without competent cells
20 立即订购
K457501
TOPO® TA Cloning® Kit for Sequencing, with One Shot® TOP10 Chemically Competent E. coli
TOP10 (genotype) 20 立即订购
K458001
TOPO® TA Cloning® Kit for Sequencing, with One Shot® TOP10 Electrocomp™ E. coli Electrocompetent TOP10 (genotype) 20 立即订购
K459501 TOPO® TA Cloning® Kit for Sequencing, with One Shot® MAX Efficiency® DH5α T1 Phage-Resistant Chemically Competent E. coli MAX Efficiency® DH5α (genotype) 20 立即订购
K453020
TOPO® TA Cloning® Kit for Sequencing, with One Shot® Mach1™ T1 Phage-Resistant Chemically Competent E. coli Mach1™ T1R (genotype)  20 立即订购
K457502 TOPO® TA Cloning® Kit for Sequencing, with One Shot® TOP10 Chemically Competent E. coli, and PureLink™ Quick Plasmid Miniprep Kit TOP10 (genotype) 20 立即订购
K457540 TOPO® TA Cloning® Kit for Sequencing, with One Shot® TOP10 Chemically Competent E. coli TOP10 (genotype) 40 立即订购
K458040 TOPO® TA Cloning Kit for Subcloning, with One Shot® TOP10 Chemically Competent E. coli Electrocompetent TOP10 (genotype) 40 立即订购
K459540 TOPO® TA Cloning® Kit for Sequencing, with One Shot® MAX Efficiency® DH5α T1 Phage-Resistant Chemically Competent E. coli MAX Efficiency® DH5α (genotype) 40 立即订购