BDB - Library Details

Library

Library Name: X15 phage display library

Library ID:

251

Library Length:

Library Complexity:

5.7e8

Library Titer:

2.5e11

Library From:

Ivone M. Takenaka

Library Randomness:

Completely random

Library Scheme:

NNM

Library Topology:

Linear

Comments:

The 15-mer phage display random peptide library was constructed using the fUSE-5 vector, kindly provided by G. Smith (University of Missouri). FUSE-5 replicative Form DNA was purified from K802 Escherichia coli and linearized by SfiI digestion, and the resulting vector DNA was ligated to synthetic DNA fragments containing complementary BglI termini. These duplex oligonucleotides contained (NNM)15 triplets in the coding strand, where N corresponds to equivalent amounts of A, T, G, and C and where M corresponds to equivalent amounts of G and T (minimizes occurrence of translational stop codons). The noncoding strand consisted of 18 or 45 residue stretches of the inosine nucleotide analog. Recombinant DNA was electroporated into MC1061 E. coli electrocompetent cells, spread on 94 plates (24 x 24 cm) with NZamine-yeast extract (NZY) agar plus 40 μg/ml tetracycline (tet) and incubated overnight at 37 °C. Phage were collected by polyethylene glycol precipitation and purified on cesium chloride gradients. The number of transducing units (TU) was determined by infecting K91Kan-resistant cells with an aliquot from each library and plating on tet plates. FUSE5-vector DNA containing an insert restores the reading frame of the pIII gene product, enabling the phage to infect K91Kan-resistant cells and produce colonies on tet plates.

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