Despite the average Kunkel mutagenesis efficiency between 50-90%, I can reach only 20% of recombinants in my pool. This is crucial moment for library construction, so I must ascend its efficiency. What can be the possible reason of it? There is no dsDNA in my uracil-containing ssDNA input DNA and no visible band of remaining ssDNA in converted double strand heteroduplex DNA. I've tried treat my double strand heteroduplex DNA with DpnI и uracil-N-glycosilase....but it didn't help