Culture wars: Empirically determining the bestapproach for plasmid library amplification

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Culture wars: Empirically determining the bestapproach for plasmid library amplification


Mateyko, N.; de Boer, C.


DNA libraries are critical components of many biological assays. These libraries are often kept in plasmids that are amplified in E. coli to generate sufficient material for an experiment. Library uniformity is critical for ensuring that every element in the library is tested similarly, and is thought to be influenced by the culture approach used during library amplification. We tested five commonly used culturing methods for their ability to uniformly amplify plasmid libraries: liquid, semisolid agar, cell spreader-spread plates with high or low colony density, and bead-spread plates. Each approach was evaluated with two library types: a random 80-mer library, representing high complexity low coverage of similar sequence lengths, and a human TF ORF library, representing low complexity high coverage of diverse sequence lengths. We found that no method was better than liquid culture, which produced relatively uniform libraries regardless of library type. However, when libraries were transformed with high coverage, culturing method had minimal impact on uniformity or amplification bias. Plating libraries was the worst approach by almost every measure for both library types, and, counter-intuitively, produced the strongest biases against long sequence representation. Semisolid agar amplified most elements of the library uniformly but also included outliers with orders of magnitude higher abundance. For amplifying DNA libraries, liquid culture, the simplest method, appears to be best.

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