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Hello people,
I have been using GeneRacer [ligase-mediated] Kit in order to amplify the 5' region of a gene of interest (~200bp). The problem is that for both initial and subsequent nested PCR the designed and kit's primers have a high Tm 76/78 oC. The polymerase i'm using is Platinum high fidelity which suggests max. 68 oC as annealing temperature and 68-72 for extension. Should I exceed the 68 as annealing and use up to 71-73 , and what would the efficient extension temp be?
Thank you a lot.
