Purification and quantitation of bacteriophage M13 using desalting spin columns and digital PCR

Separation of small molecules such as biotinylated baits from solutions of filamentous bacteriophage is achieved generally through polyethylene glycol precipitation of the phage and centrifugation prior to affinity selection or panning. This method is laborious and time-consuming and is accompanied frequently by significant loss of virions, especially when performed at low phage concentrations. Similarly, accurate quantitation of phage is performed typically by counting plaques, a method that is tedious, low-throughput, and not amenable easily to high titers. In this report it is demonstrated that commercially available Zeba Spin Desalting Columns are useful devices for the efficient separation of small molecules from bacteriophage, which pass through almost unimpeded and remain infectious. It is shown further that digital PCR on microfluidic chips is a fast and accurate high-throughput technique to determine phage genome concentrations precisely. (C) 2012 Elsevier B.V. All rights reserved.