Topo cloning how does it work

Salt helps prevent topoisomerase I from rebinding, which results in more intact molecules. Note that the amount of salt you add will depend on whether you are planning on transforming your reaction into chemically or electro-competent E. Shuman S.

Novel approach to molecular cloning and polynucleotide synthesis using vaccinia DNA topoisomerase. J Biol Chem. Topics: Plasmids , Plasmid Cloning , Plasmids. Add Comment. Addgene is a nonprofit plasmid repository. We archive and distribute high quality plasmids from your colleagues.

You must be logged in to post a comment. This site uses Akismet to reduce spam. Learn how your comment data is processed. Facebook Twitter LinkedIn More. Written by Dr Rebecca Tirabassi. Leave a Comment Cancel Reply You must be logged in to post a comment. There is no need to manually add these modifications as Geneious will detect the correct modifications automatically.

The only requirements are that you have a circular vector containing a TOPO site and a compatible insert.

The green icon associated with this file indicates that the sequence is circular. As this motif is palindromic it will be found and annotated on the forward and reverse strands. Uncheck the option for Forward strand so that the motif is only found and annotated onto the reverse strand of the vector. Zoom into position bp and you will see the new motif which is located between two Eco RI restriction sites.

In practise TOPO-cloning is not directional, so the insert is equally likely to ligate in both directions. However, in Geneious only the current orientation defined by the orientations of documents in the sequence viewer will be ligated into the vector. If you should need both insert orientations, create a copy of the insert, make a reverse complement use the R. We want this promoter to be oriented in the same direction as the Human Proinsulin sequence which is oriented in the forward direction , so that we can eventually use it to express our gene product.

Thus, we should reverse the vector sequence before we proceed by clicking on R. Choose to reverse complement entire sequence , then click OK and then Save. Now select the Human Proinsulin sequence.