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HomeFAQsI am using Ph.D.™ Phage Display and after 4 or more rounds of panning all clones are wild-type phage (white plaques).
FAQ: I am using Ph.D.™ Phage Display and after 4 or more rounds of panning all clones are wild-type phage (white plaques).
In a typical round of biopanning, ~2 x 1011 input phage are reacted with the target, and between 103 and 107 total phage are eluted off following washing. This corresponds to an enrichment of 104 to 108-fold per round. Since the library contains ~2 x 109 different clones, the eluted pool of phage should in theory be fully enriched in favor of binding sequences after only 2 or 3 rounds. Once this point is reached, further rounds of amplification and panning will result only in selection of phage that have a growth advantage over the library phage. For example, vanishingly small levels of contaminating environmental wild-type phage (less than one part per billion) will completely overtake the pool if too many rounds of amplification are carried out, regardless of the strength of the invitro selection.