Note that some enzymes need a certain number of nucleotides beyond their recognition site for digestion. They found that proteinase K treatment increased the yield of their clones. Vectors are commercially available that have an overhanging T on them to compensate for the potential overhanging A at the end of some PCR products. We have had many successful ligations of PCR products into blunt-end digested vectors, suggesting that there is a significant proportion of molecules that lack the overhanging A residue. The method does not use ligation, and reportedly produces only recombinants with high efficiency.
Small inserts can give light blue colonies when a pUC-based vector is employed.
Other parameters have also been found that influence this measurement. To maximize sequence fidelity of the products, or for use in random mutagenesis, the following table may be helpful. Reducing it may help. Increase the ratio of template to primers. Reduce the number of cycles.