b'PCR cloning PCR cloning is a method in which double-stranded DNA fragments amplified by PCR are ligated into a vector. With PCR amplification, this cloning technique requires much less starting material for the insert sequence and allows introduction of new restriction and/or recombination sites to the 5 end of the inserts.TOPO cloning Gateway cloningTOPO PCR cloning technology was developed to help improve cloning efficiency,To shuttle a PCR insert among vectors, the Gateway cloning system offers site-specific, simplify protocol setup, and accommodate a wide range of PCR insert sizes. TOPOrecombinase-based cloning. It maintains the inserts proper orientation and reading cloning vectors are linearized by the activity of topoisomerase I (which also has a ligaseframe during shuttling using the Gateway vectors. Once a gene is cloned into an function) that is covalently bound to the 3 phosphate on each end (see figure below).entry clone, you can then move the DNA fragment into one or more destination This system enables the vectors to be joined to PCR inserts with compatible ends (withvectors simultaneously.up to 95% efficiency), without the need for additional ligation steps, in 5 minutes. Find out more at thermofisher.com/gatewayFind out more at thermofisher.com/topoAdd 1 L of PCRPerform transformation Perform PCR with Taq orreaction to 1 L ofIncubate 5 minutes at with provided competent proofreading polymerase TOPO cloning vector room temperature E. coli strainA PCR product A PCR productCACC PCR productTOPOTOPOTOPOcloningvectorTOPO PCR cloning requires just three easy steps.Quickly find your TOPO cloning kit with our new interactive selection tool. Search by Cloningapplication, vector, or desired competent cells at thermofisher.com/topoguideDid you know?The Invitrogen TOPO XL-2 Complete PCR Cloning Kit provides all the necessary elements for highly efficient cloning of extra-long PCR products from 113 kb. thermofisher.com/topoxl2 42 Contents'