Phagemid libraries

Add 100 pi of glycerol library stock to each of two 1 litre flasks containing 500 ml of 2TYG with 100 pg/ml ampicillin. Grow at 37 C with rapid shaking (300 r.p.m.) for 1-2 h or until A oo is between 0.5-1.0. 

Add M13K07 to a final concentration of 5 x 10 plaque forming units (p.f.u.)/ml. This should give a multiplicity of infection (m.o.i.) of approximately 10. 

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Take 40 mL of the 50-mL culture (primary phagemid library stock grown as described in Section 3 3.1.), and centrifuge down the bacteria at 3300g for 10 min. 

O Connel, D., Becerril, B., Roy-Burman, A., Daws, M., and Marks, J. D. (2002) Phage versus phagemid libraries for generation of human monoclonal antibodies. J. Mol. Biol. 321,49-56. 

In conclusion, these feasibility exercises were successful, but I am presently unaware of their use in actual library construction or selective panning. The alternative system for cytoplasmic expression, which has been well characterized in generating gene banks and panning selection, is the plasmid-protein-complexlisplay system (also called peptides-on-plasmids ) developed by Schatz [127-130] using extensions to the C-terminus of the lac-repressor. This is not a phagemid system, since the plasmid DNA is naked and is reintroduced into the cell by transformation. 

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