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Trộn xoáy, khai thác, sau đó quay một thời gian ngắn trong microcentrifuge. 6. Ủ các ống ở 72 ° C trong 2 phút, sau đó mát mẻ trong khoảng thời gian 60 phút đến dưới 30 ° C. Lưu trữ các ống trong nước đá hoặc ở nhiệt độ 4 ° C. 7. Thêm thuốc thử phản ứng ủ như trong Bảng 2. 8. Trộn xoáy, khai thác, sau đó quay một thời gian ngắn trong microcentrifuge. | 278 Chowdhury Table 2 Synthesis of the Mutagenic Strand Experimental pL Control pL 10X Synthesis buffer 1.3 1.3 T4 dNa ligase 3 U pL 1.0 1.0 T7 DNA polymerase 1.0 1.0 1U pL diluted 1 1 in T7 polymerase buffer Total 13.3 13.3 5. Mix by tapping vortex then spin briefly in a microcentrifuge. 6. Incubate the tubes at 72 C for 2 min then cool over a period of 60 min to less than 30 C. Store the tubes in ice or at 4 C. 7. Add reagents to the annealing reactions as in Table 2. 8. Mix by tapping vortex then spin briefly in a microcentrifuge. 9. Incubate in ice for 10 min then at room temperature for 10 min and finally at 37 C for 2 hours. Store in ice. 10. Transform 50 pL competent TG1 cells with 1 pL reaction mix and plate 10 25 50 and 100 pL onto LB agar plates containing 2 glucose and the appropriate antibiotic for selecting the scFv-glllp-encoding phagemid. Incubate overnight at 37 C. Pick and analyze clones for introduction of the TAA mutation. 3.2.5. Introduction of Random Mutations at Selected Hotspots Construction of the randomized library can also be done by Kunkel s mutagenesis see Note 9 using as template the scFv-glllp-encoding phagemid with introduced TAA stop codon see Note 10 . Preparation of ssuDNA has been described see Subheading 3.2.1. . 1. The basic rules for designing oligonucleotides are as described in Subheading 3.2.3. but in the present case one must keep in mind the following points Codons falling into the hotspot regions have to be randomized the region to be randomized must encompass the TAA stop codon introduced in Subheading 3.2.4. if the TAA stop codon was inserted outside the hotspot region then it must be changed to the original codon or one encoding the wild-type amino acid by adding an additional oligonucleotide to the annealing mix see Subheading 3.2.4. steps 1-6 . The oligonucleotides to be used for randomization should have degenerate codons such as NNS N is A G C or T S is G or C which codes for all the amino acids but not the TAA and
