Annealing temperature :)

but can you give me some advices, is the first time in use of this polimerase and i want to make all the calculations for the PCR conditions and concentrations thx!

but you controle the Mg concentration only decreasing the Buffer HF 5X concentration or you have different technique :)

may be this article could help you:

Breslauer, K J., Frank, R., Blöcker, H., Marky, L A., (1986) Predicting DNA duplex stability from the base sequence. PNAS 83 (11), 3746-50

Antonio Gracias! lo leere de inmediato , Brad i have a few questions and a image of the gradient temp if u can help me thanks

Hi! Can you please give me the name of your enzyme? I´m trying to amplify 2400 bp but I can´t. I´m using High Fidelity and Pfu. Please, help me! Thanks :)

claro, Phusion Polymerase i sent you the link of the catalogue http://www.fermentas.com/en/products/all/pcr-qpcr-rt-pcr/high-fidelity-pcr/f530-phusion-high-fidelity-dna-polymerase

All calculations of annealing temperatures are more or less heuristic and will sometimes fail to predict the optimum annealing temperatures. If you do a specific PCR often, optimization (using a gradient PCR) can be performed. In such a PCR, the annealing temperature of the block is different for adjacent tubes and thus you can e.g. test a range of annealing temperatures (e.g. from calculated Tm-10 to calcualted Tm+10) to find the best one. However, not all PCR machines support gradients. There seem to be also differences between polymerases and some apparently tolerate incorrectly chosen annealing temperatures better than others. The one that seems to be best in this respect in our hands is the KAPA polymerase (see here: https://jeltsch.org/PCR_polymerase_comparison). You can get yourself a free sample to test it if you have problems with Phusion or any other polymerase. 

https://jeltsch.org/PCR_polymerase_comparison