Directional TOPO cloning enables cloning of blunt-ended PCR products in a 5´→3´ orientation directly into a expression vector using a 5-minute ligation reaction, thereby eliminating subcloning steps and saving you time.
Directional TOPO cloning vectors contain a single-strand GTGG overhang on the 5´ end and a blunt end on the 3´ end. The four-nucleotide overhang invades the double-strand DNA of the PCR product and anneals to the CACC sequence that you place in your 5´ primer. Topoisomerase I then ligates the PCR product in the correct orientation.
With Directional TOPO Cloning Expression Kits, you will:
- Save time – TOPO cloning of your PCR product takes just five minutes
- Obtain efficient cloning results – recombinant clones (>90%) will be in the correct orientation for expression (Figure 1)
- Achieve high-level expression – vectors carry powerful promoters for expression in E coli or mammalian cells
Figure 1. Directional cloning of human open reading frames into pcDNA 3.1 D/VS- His- TOPO vector.
Resources
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