BDB - Library Details

Library

Library Name: X8 M13 phage display library

Library ID:

336

Library Length:

Library Complexity:

Library Titer:

Library From:

Angela M. Belcher

Library Randomness:

Completely random

Library Scheme:

NNM

Library Topology:

Linear

Comments:

The M13KE phage vector was modified by making a cloning site for pVIII display. A Pst I restriction site was made by mutating T to A at position 1372, a BamH I site was made by mutating C to G at positon 1381, and the Pst I site at position 6246 was deleted by mutating T to A at position 6250. The site-directed mutagenesis was done using overlap extension PCR. A dsDNA library was then prepared and cloned into the resulting modified phage vector, named M13SK, using Pst I and BamH I. To obtain the dsDNA library, partial library duplexes were formed by annealing of extension primer (5\'-GATGCTGTCTTTCGCTGCAG-3\') with oligonucleotides (3\'-ACGACAGAAAGCGACGTCnm(nnm)6nnCCTAGGAACATC ATC-5\', where n=A, T, C, or G and m=A or C). The partial library duplexes were incubated with Klenow fragment (3\'→'5\' exo-) (10 U/μL) and dNTP at 37 °C for 30 min. The Klenow fragment was inactivated by heating (75 °C for 20 min), and the mixture was digested with Pst I and BamH I. The digested DNA was gel purified (2-40% TBE polyacrylamide gel), ligated into M13SK, and transfected to XL1-Blue Electroporation Competent Cells using a MicroPulser (Biorad). The library was titered according to manufacturer directions and sequenced (MIT Biopolymers Laboratory) before amplification.

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