Team:Heidelberg/Templates/Del Methods 10


Gibson Cloning is all about designing the most suitable overlap for both amplification of your desired genes and ligation of the final fragments. Overlaps ought to be long enough to ensure specifity in annealing with its complementary region of another fragment. Nevertheless that doesn't mean that one can design overlaps of arbitrary lenght. You have to consider the annealing temperautre of 50°C at which the Gibson Assembly is conducted as well as possible secondary structes. Use mFold to avoid unexpected issues. You can easily avoid problematic secondary structues by taking care of the GC-content. Adjusting GC content between 45 and 55% significantly improved formation of secondary structures and therefore amplification from the genome of our donor organism. For us overlaps of 20 to 40 basepairs in total prooved to be very effective. These numbers have to be increased, in case you introduce promotors or ribosome binding sites with your primers