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5 years ago

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Hot start PCR using phusion green DNA polymerase

I am currently performing hot start PCR using phusion green hot start DNA polymerase in order to amplify a 8.8kb insert. However, I cannot get any amplified product at all. I have tried changing some parameters but I am not sure what to do. Any help?

Experiment:

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Make sure your PCR cycles are correct. After that you should try to optimize your reaction with the addition of DMSO and by adjusting the MgCl2 concentration. Basically, run multiple reactions and adjust MgCl2 in 0.2-1 mM increments. One last thing that might be affecting your PCR is the GC content. You can try adding a GC buffer or enhancer to your PCR mix.

Answered 5 years ago

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