The vector used to demonstrate PFunkel mutagenesis here is a low-copy phagemid containing the p15a origin. The preparation of ssDNA template using this phagemid as described results in a mixture of m13 helper phage ssDNA and phagemid ssDNA, in which the m13 helper ssDNA is the more abundant species. The indicated 1 ug of ssDNA template added to the PFunkel reaction represents a mixture of the two species. We therefore recommend that when performing PFunkel on higher-copy phagemids (in which the prepared ssDNA will contain a higher fraction of phagemid ssDNA), it may be necessary to reduce the amount of template added. This clarification does not apply for performing PFunkel on a dsDNA template.

-Marc Ostermeier