We are working with a nonconventional yeast that has a GC content in 20% range. While I have worked with high GC content organisms these low GC organisms are offering a special difficulty, especially with UTR primers. When designing primers I will have very low melt temps with long stretches that also have very low delta G hairpins and primer dimers. We often get nonspecific priming or no amplification at all. I have played with melting temps, using purified genomic DNA (which is cumbersome as we are working on screens), and adding dmso but have seen no real improvement. We are using NEB Q5 pol.