Rapid isolation of highly pure single-stranded DNA from phagemids.

Single-stranded DNA (ssDNA) has many applications in molecular biology and biotechnology. The conventional method for the preparation of ssDNA from phagemids is laborious, costly, and inefficient. Here we describe an integrated protocol for consistent production of phagemid ssDNA from a bacteria/phagemid/help phage complex and rapid isolation and purification of the ssDNA with a silica column followed by duplex-specific nuclease digestion. The major advantages of our method are the expediency, low cost, and consistent yield of highly pure ssDNA that is suitable for direct sequencing and other applications. This method is especially useful for large-scale preparation of high-quality ssDNA.