ZeroBlunt® TOPO® PCR Cloning Kits

TOPO® cloning technology removes time-intensive and unreliable steps from your Blunt end cloning workflow, allowing you to perform benchtop cloning reactions in just 5 minutes. With up to 95% recovery of your desired clone, you always have the clone you need for downstream experiments.

With more than 10 years of established performance and over 4,000 scientific citations, TOPO® Blunt end cloning is the method of choice for researchers around the world.

Three simple steps save time

TOPO® PCR cloning requires just three easy steps.

  1. Combine your PCR product and a TOPO® cloning vector in the provided solution
  2. Wait 5 minutes
  3. Transform E. coli

With TOPO® Cloning the additional time, steps, and reagents required for ligase-mediated cloning are eliminated.

Zero Blunt® TOPO® PCR Cloning Kits

Invitrogen's Zero Blunt® TOPO® PCR Cloning Kits combine our unique Zero Background™ technology (see box below) with TOPO® Cloning in the pCR®-Blunt II-TOPO® vector (Figure 1). Together these enable easy, high-efficiency DNA cloning of blunt-end PCR products produced using high fidelity PCR enzymes.


Convenient features of this DNA Cloning vector include:

  • ccdB gene to eliminate background
  • EcoR I sites flanking the PCR product insertion site for easy removal of inserts
  • Kanamycin and Zeocin™ resistance genes for your choice of selection in E. coli
  • T7 and SP6 sites for in vitro RNA transcription
  • M13 forward and reverse primer sites for sequencing or PCR screening
  • Selection of competent cells for high speed, electroporation, or routine cloning

Figure 1: Convenience, Ease of Use, High Copy. The pCR® - Blunt II TOPO® vector has convenient validated restriction sites for easy subcloning, dual selection markers for E. coli expression and a pUC origin that enables recovery high yields of plasmid.

Eliminate high background

Because of high background, cloning blunt-end and long PCR products can be difficult and often yields a low percentage of recombinants. Invitrogen’s unique Zero Background™ technology uses the lethal ccdB (control of cell death) gene to enable high-efficiency cloning, yielding nearly 100% recombinants.  The ccdB protein poisons bacterial DNA gyrase, causing degradation of the host chromosome and cell death.1,2 When an insert is ligated into the vector, the ccdB gene is disrupted, enabling only recombinant colonies to grow (Figure 2).


Figure 2:  Zero Background™ technology enables recovery of only recombinant clones.

References

  1. Bernard, P. and Couturier, M. (1992) J Mol Biol 226: 735–745.
  2. Bernard, P. et al. (1993)  J Mol Biol 234: 534–541.
  3. Bernard, P. et al. (1994) Gene 148: 71-74.