cDNA Long Essay for BIO325 Exam 2

cDNA Long Essay for BIO325 Exam 2 - cDNA Long Essay for...

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cDNA Long Essay for BIO325 Exam 2 Making a cDNA Library 1. Isolate all total mRNAs from cells 2. Reverse transcriptase mRNAs to make ssDNAs, then use DNA pol to make complementary DNA (cDNA) to the mRNA 3. Cut ends of cDNAs with restriction endonucleases to give them sticky ends 4. Ligate cDNAs into cosmid vectors with same restriction endonucleases used in step 3 in order to make the sticky ends match. Cosmids are used because they can handle an insert of 46 kb 5. Package recombinant cosmids into capsids, which are self-packaging that encase DNA into phages 6. Infect bacteria with cosmid particles, and plate bacteria as a lawn to grow more cosmids to reproduce inside cells. The bacteria, when spread as a lawn, are spread densely to grow a solid opaque layer when grown. Plaques are clear spots on the lawn when grown and are live bacteria representing a different cDNA. 7. Overlay plate with nylon to have DNA and cosmid particles to stick to
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