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Antibody Phage Display Methods and Protocols - part 4

Superinfect với VCSM13 thể thực khuẩn helper với tỷ lệ 20 1 thể thực khuẩn vi khuẩn và chuẩn bị thể thực khuẩn như lập chỉ mục ở những nơi khác trong khối lượng. Ngoài ra xác định tỷ lệ cfu / PFU mạ pha loãng nối tiếp của các thể thực khuẩn trên tấm | 108 Sharon et al. 10. Repeat steps 7 and 8 for phh3-VL-VH-lib see Fig. 5 and store in aliquots at -80 C as the original VL-VH library stock. 11. Plate an aliquot of the VL-VH library 1 X 109 bacteria for a library of 1 X 107 members at high density on LB-CARB-GLU plates. Scrape the bacterial colonies and grow in LB-CARB-GLU containing 10 pg mL tetracycline LB-CARB-GLU-TET to an OD600 of 0.5. Superinfect with VCSM13 helper phage at a 20 1 ratio of phage bacteria and prepare phage as indexed elsewhere in this volume. Also determine the cfu pfu ratio by plating serial dilutions of the phage on LB-CARB plates for phagemid colonies and on B plates per L 10 g Bacto-tryptone 8 g NaCl 15 g agar for plaques a cfu pfu ratio 1 is desirable . 12. Carry out positive negative selection on the phage following appropriate methods. See index for details. 13. After positive negative selection infect XL1-Blue supercompetent bacteria with the selected phage. Plate the infected cells on LB-CARB-GLU plates in serial dilutions to determine the size of the selected library and at high density to recover the library. Scrape the bacterial colonies and superinfect a portion of the culture with VCSM13 helper phage to produce phage for immunoassay e.g. enzyme-linked immunosorbant assay against the poly-Ag target . Store the remainder of the selected library culture in aliquots in LB-CARB-GLU-15 glycerol at -80 C. 14. Prepare dsDNA from the selected library and digest with SacI and XhoI. Gel-purify the 5-kb backbone see Note 4 . Also isolate the 1.8-kb SacI XhoI fragment from vector no. 578 plPEHPl see Fig. 6 the bidirectional mammalian lPPl cassette which carries the mammalian promoter and leader sequences and the mouse Ig p enhancer. Ligation of these fragments will generate phh3-VL-m-VH-lib. 15. Transform phh3-VL-m-VH-lib into supercompetent HB101 cells and plate on LB-CARB plates in serial dilutions to determine the size of the selected library and to ascertain that 90 of library members .