BDB - Library Details

Library

Library Name: pVIII fth1-dp-based X7 phage display library (X7)

Library ID:

466

Library Length:

Library Complexity:

Library Titer:

Library From:

Jonathan M. Gershoni (Tel Aviv University, Tel Aviv, Israel)

Library Randomness:

Completely random

Library Scheme:

NNK

Library Topology:

Linear

Comments:

The fth1-dp vector is a derivative of the fth1 vector. To adapt the fth1 for Illumina deep sequencing, Illumina adaptors A and B were inserted upstream and downstream to the insert-flanking SfiI sites. Oligonucleotides corresponding to the Adaptor sequences were inserted by 'SOEing' PCR mutagenesis using the Accuzyme polymerase (Bioline, BIO-21052). Alternatively, extended PCR primers can be used to accomplish the same as has been described previously. Libraries were constructed as described below. Briefly, two 5’ biotinylated oligonucleotides were used. The first contained the redundant "library" sequence, e.g., 7×NNK flanked by BglI sites compatible with the two SfiI sites of the vector (61 bases). The second oligonucleotide, 18 bases, complemented the 3’ end of the first and was extended to "fill-in" the complementary strand using Klenow polymerase. The product was digested with BglI, the short biotinylated segments were removed with Streptavidin conjugated magnetic beads and the eluent was cloned into SfiI digested fth1-dp vector. This ligation mix was used to electroporate MC1061 or DH5alpha cells as indicated in the text.

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