Highly efficient, 5-minute cloning with TOPO

TOPO TA in vitro transcription kits utlize the pCRII-TOPO TA vector with T7 and SP6 promoters for in vitro RNA transcription and sequencing. TOPO TA technology works by directly ligating Taq-amplified PCR products in a 5-minute, benchtop reaction yielding up to 95% recombinants.

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pCRII-TOPO TA vector diagram

The pCRII-TOPO TA vector features:

  • 3´-T overhangs for direct ligation of Taq-amplified PCR products
  • T7 and SP6 promoters for in vitro RNA transcription and sequencing; the vector also contains M13 forward and reverse primer sites for sequencing
  • EcoRI sites flanking the PCR product insertion site for easy excision of inserts
  • Kanamycin and ampicillin resistance genes for your choice of selection in E. coli
  • Easy blue/white colony screening for selection of recombinants

The pCRII-TOPO TA vector

Performance and value

Each TOPO TA Cloning for in vitro transcription kit contains linearized and topoisomerase I-activated pCRII-TOPO 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.

Using a proofreading enzyme?

Thermostable polymerases containing extensive 3´ to 5´ exonuclease activity, such as Platinum SuperFi, 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.

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