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21st Century Manufacturing Episode 2 Part 11

Tham khảo tài liệu '21st century manufacturing episode 2 part 11', kỹ thuật - công nghệ, cơ khí - chế tạo máy phục vụ nhu cầu học tập, nghiên cứu và làm việc hiệu quả | 394 Biotechnology Chap. 9 h . 1 H . fc- z z it Plasmid DNA .n p n . Mix D andenz 1 Incubate 37 c MAs Plasmi me tn am D p lHeat to60 c then cool VA cut Endonuclease gene inactivated Add---- Recombinant---J DNA 1 DNA i Transformed----- . coli cells I Agar contains ampicillin E Agar contains Two pure cultures containing tetracycline cloned rabbit DNA Figure 9M Procedures for obtaining pure clones containing rabbit DNA a plasmid DNA with resistance to tetracycline and ampicillin is mixed with rabbit cDNA b DNA ligase is added to join the DNA fragments c E. coli cells act as host d plasmid-containing cells are selected by growth on agar containing tetracycline and cells with plasmids joined to rabbit cDNA are identified by screening on ampiciilin-containing agar e and f these cells grow only on tetracycline g pure cultures containing cloned genes from Understanding DNA and Gene Cloning by Drlica Copyright 1992. Reprinted by permission of John Wiley Sons Inc. . 9.9 Bioprocess Engineering 395 The cDNA could then be isolated made single-stranded typically by boiling and radioactively labeled. 9.8.7 Step 5 Final Screening by Nucleic Acid Hybridization to Isolate Genomic Clones Bottom of Figure 9.23 The final step is to screen the colonies grown from the phages containing the original fragmented rabbit DNA and to find which ones have the desired hemoglobin genes recall this is on the right side of Figure 9.23 .The DNA in each plaque is tested to see if it will hybridize with the radioactive hemoglobin cDNA nucleic acid complement to the hemoglobin genes being sought.The technique relies on the principle of complementary base pairing between the cDNA probes and the desired hemoglobin genes in those very few plaques described in Step 2. Practical Techniques After the phages were used to grow E. coli colonies on an agar plate a piece of filter paper was placed on the agar and removed. The cells were thus transferred to the paper which was placed in a dilute solution of sodium .