The slowest-running band is the strand-displaced DNA that arises from inherent strand-displacement activity of T7 DNA polymerase and has a low mutation rate (around 20%)23. design, library construction, hybridoma technology immune response for mAb production5. In 1978, Hutchison reported the use of an oligonucleotide to direct mutagenesis of a residue in a single-stranded bacteriophage computer virus6. In 1985, Smith reported that foreign gene fragments can be fused in frame with the gene encoding phage coat protein III and can thus be displayed around the phage surface without compromising its infectivity7. These pioneering works laid a foundation for the subsequent construction of phage-displayed antibody libraries in immune, na?ve, and synthetic forms with the formats of single-chain variable fragment (scFv) and antigen-binding fragment (Fab) for therapeutic mAb development8,9. From the technical point of view, phage display-based antibody development offers a complementary approach to hybridoma-based mAb development that can help to circumvent the limitations some antigens can pose and the humanization process that hybridoma-derived antibodies often require5. As of 2016, 6 phage display-derived mAbs have been approved in the market including Humira, one of the most successful mAbs used for treatment of rheumatoid arthritis, and many phage display-derived antibody candidates are currently at various stages of clinical investigation10. Lanopepden For immune and na?ve phage antibody libraries, the diversity of complementarity-determining regions (CDRs) in light and heavy chain is derived from the natural immune repertoire (by electroporation. For high diversity, phage-displayed library construction, high-voltage electroporation of a two-component mixture of electro-competent cells and covalently closed circular dsDNA (CCC-dsDNA) should be prepared carefully. Sidhu altered the preparation of electro-competent cells and DNA from traditional methods and greatly improved library diversity14. In this protocol, we describe a method for construction of synthetic phage-displayed Fab libraries with diversities of 109-1010 obtainable with a Lanopepden single electroporation. Physique 1 shows an overview of library construction including: 1) high-efficiency electro-competent cell preparation; 2) extraction of dU-ssDNA; 3) Kunkel’s method based oligonucleotide-directed mutagenesis; 4) electroporation and calculation of library size; 5) protein A/L-based ELISA for folding and functional diversity evaluation; and 6) DNA sequence analysis of diversity. All the reagents, strains and gear are listed in the Lanopepden Material’s Table. Table 1 shows the reagent setup. Open in a separate window Protocol NOTE: Filter sterile tips Lanopepden must be used throughout when dealing with phage to avoid contamination to pipette gun and surrounding area. Aseptic area or hood must be used when handling with bacteria and phage experiments. Phage experiment area must be cleaned up using 2% sodium dodecyl sulfate (SDS) followed by 70% ethanol to avoid phage contamination. For making serial dilutions in this protocol, new tips should be used for each dilution. 1. SS320 Electro-competent Cell Preparation Pre-warm LB/tet agar plate (prepared and stored at 4 C for less than 1-week aged) at 37 C incubator for 1 h. Use a sterile inoculation loop or sterile tip to streak out a glycerol stock of CJ236 made up of Fab backbone phagemid to inoculate a starter culture of 3 mL 2YT/carb/cmp in a 14-mL polypropylene round-bottom tube, and grow at 37 C with shaking at 200 rpm overnight for around 12 h. Inoculate the 0.3 mL starter culture into 30 mL 2YT/carb/cmp in a 250-mL baffled bottle. Incubate the cell culture at 37 C LEFTY2 with shaking at 200 rpm for 3-4 h until OD600 is usually reached at around 0.4-0.8 (log phase growth). Add the M13KO7 (lab stock, titer of approximately 1 x 1013 cfu/mL) to the culture from step 2 2.8 with a multiplicity of contamination (MOI) of approximately 10 and the final titer of M13KO7 is?approximately 1 x 1010 cfu/mL. Incubate at 200 rpm and 37 C for.