Directional TOPO Cloning Topoisomerase I recognition sites Topoisomerase I 1.Cleaves and linearizes the pENTR vector, allowing insertion of the PCR fr
{"Image DocumentName": "Directional TOPO Cloning", "Image ImageDescription": "Topoisomerase I recognition sites. Topoisomerase I. Cleaves and linearizes the pENTR vector, allowing insertion of the PCR fragment. Directional joining of double-stranded DNA using TOPO\u00ae-charged oligonucleotides occurs by adding a 3\uf0a2 single-stranded end (overhang) to the incoming DNA. This single-stranded overhang is identical to the 5\uf0a2 end of the TOPO\u00ae-charged DNA fragment. At Invitrogen, this idea has been modified by adding a 4 nucleotide overhang sequence to the TOPO\u00ae-charged DNA. In this system, PCR products (up to 5 kb) are directionally cloned by adding four bases to the forward primer (CACC). The overhang in the cloning vector (GTGG) invades the 5\uf0a2 end of the PCR product, anneals to the added bases, and stabilizes the PCR product in the correct orientation. Inserts can be cloned in the correct orientation with high efficiency. 2. Ligates the vector.", "Image XResolution": "37", "Image YResolution": "37", "Image ResolutionUnit": "Pixels/Centimeter", "Image Software": "SlidePlayer", "Image YCbCrPositioning": "Centered"}