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.

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 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