Team:Heidelberg/Templates/Del Methods 11


When dealing with Gibson primers and very long amplicons, sometimes amplifications turn out to be quite complicated. These issues occure especially when one primer has a normal lenght of about 18-26 bp while the other one is a Gibson primer of about 100 bp introducing a promotor and/or a ribosome binding site. In that case the differnce between the primers annealing tempreatures most certainly will be higher than 15°C. Furthermore complex secondary structues of the Gibson primer will most likely require high temperatures at which the ordinary primer will not anneal anymore. Complex sequences such as the one of the Delftibactin cluster from D. Acidovorans displaying a extraordinary high GC content make it even harder to yield high amounts of specific product as not only the primers but also the template creates secondary structures inhibiting elongation. Nevertheless some constructs especially NRPS coding sequences require such PCRs to be carried out. In order to optimize the procotols for such complex conditions it is very crucial to use a long distance high fidelity polymerase. Obviously most optimization can be done by investigation of the optimal annealing temperature. This is also to be taken in consider when designing your primer pairs. Touchdown PCRs are a very useful tool for the described amplicons. Therefore the procedure of a touchdown PCR will be explained in week XY Nevertheless there was one agent which by the end was used in all our amplifications with the Phusion flash polymerase since it turned out to be the magic potion for our PCRs. DMSO interferes with the selfcomplementary of both, template and primers and therby inhibts formation of secondary structures. However one has to take in account, that addition of 5% DMSO decreases the annealing temperatures of primers by 3 to 5 °C. Last but not least, primer concentration it very crucial for successfull amplification. High primer concentrations is very specific reaction lead to significantly increased yield. Whereas high primer concentrations in reactions of low specifity or in cases in which primers do not bind at al favor formation of primer dimers and thereby are inhibitory for the reaction.