BDB - Library Details

Library

Library Name: LAP yeast display library

Library ID:

334

Library Length:

Library Complexity:

Library Titer:

Library From:

Alice Y. Ting

Library Randomness:

Semi-random

Library Scheme:

Library Topology:

Linear

Comments:

The LAP library is displayed on the yeast surface as a fusion to Aga2p protein. A C-terminal myc epitope is used to quantify LAP expression level. A partially randomized oligo with the following sequence: 5'A AAT AAG CTT TTG TTC GGA TCC NGM MNN NAN NTS MNN MNN AAC TTT ATC MNN NTS NAN TCC GCT AGC CGA CCC TCC, was ordered from IDT (Integrated DNA technologies). N designates an equimolar mixture of all bases. S designates a 1:1 mixture of G and C. M designates a 1:1 mixture of A and C. This oligo was annealed with another oligo, Con2For.F (5'CT AGT GGT GGA GGA GGC TCT GGT GGA GGC GGT AGC GGA GGC GGA GGG TCG GCT AGC GGA), which overlaps with both pCTCON2 vector and the library oligo. The 5' overhangs were filled in using Klenow polymerase. The resulting product was PCR-amplified using the primers Con2For.F and Con2Rev.R (5'TA TCA GAT CTC GAG CTA TTA CAAGTC CTC TTC AGA AAT AAG CTT TTG TTC GGA TCC). Meanwhile, pCTCON2 vector was prepared by digestion with NheI and BamHI, and gel-purified. PCR insert and pCTCON2 vector were transformed together into S. cerevisiae EBY100 (Invitrogen) by electroporation as described by Colby et al.52 Homologous recombination occurred inside the yeast. Serial dilutions of transformed yeast were plated on SDCAA plates and colonies were counted, to determine transformation efficiency.

First |

Prev |

334

| Next

| Last