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Plasmid (BAC) Phagemid Cosmid Lambda (PAC)

Lots of tools for cloning:. Plasmid (BAC) Phagemid Cosmid Lambda (PAC). Needs to be at least 40kb to grow, but A left and right arm can be made smaller… See next slide. Intron and Exon in Eukaryotic Cells. exon. exon. exon. intron. intron. 5’. 3’. DNA. promotor. 3’. 5’.

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Plasmid (BAC) Phagemid Cosmid Lambda (PAC)

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  1. Lots of tools for cloning: Plasmid (BAC) Phagemid Cosmid Lambda (PAC)

  2. Needs to be at least 40kb to grow, but A left and right arm can be made smaller… See next slide

  3. Intron and Exon in Eukaryotic Cells exon exon exon intron intron 5’ 3’ DNA promotor 3’ 5’ stop codon start codon Transcription mRNA Processing cap poly A tail Splicing Intron deleted mature mRNA To cytoplasm Take place in nucleus

  4. AG/GT CAG/NT exon 1 intron 1 exon 2 Eukaryotes Splice Signals

  5. cDNA Is Reverse Transcribed from mRNA 5’ 3’ 3’ 5’ 5’ Baltimore, Dulbecco, Temin (1975) mature mRNA poly A tail Reverse transcription TTTT 3’ 5’ RNA hydrolysis DNA polymerase 3’ CCC 5’ GGG 3’

  6. cDNA Library Genes in expression mRNA Reverse transcription Complete gene cDNA Vector: Plasmid

  7. Libraries: cDNA cDNA library has sequences only from genes transcribed into mRNA (~3% of genome)

  8. MNGTEGPNFYVPFSNKTGVVRSPFEAPQYYLAEPWQFSMLAAYMFLLIML GFPINFLTLYVTVQHKKLRTPLNYILLNLAVADLFMVFGGFTTTLYTSLH GYFVFGPTGCNLEGFFATLGGEIALWSLVVLAIERYVVVCKPMSNFRFGE NHAIMGVAFTWVMALACAAPPLVGWSRYIPEGMQCSCGIDYYTPHEETNN ESFVIYMFVVHFIIPLIVIFFCYGQLVFTVKEAAAQQQESATTQKAEKEV TRMVIIMVIAFLICWLPYAGVAFYIFTHQGSDFGPIFMTIPAFFAKTSAV YNPVIYIMMNKQFRNCMVTTLCCGKNPLGDDEASTTVSKTETSQVAPA*

  9. Maxim-Gilbert Sequencing MNGTEGPNFYVPFSNKTGVVRSPFEAPQYYLAEPWQFSMLAAYMFLLIML GFPINFLTLYVTVQHKKLRTPLNYILLNLAVADLFMVFGGFTTTLYTSLH GYFVFGPTGCNLEGFFATLGGEIALWSLVVLAIERYVVVCKPMSNFRFGE NHAIMGVAFTWVMALACAAPPLVGWSRYIPEGMQCSCGIDYYTPHEETNN ESFVIYMFVVHFIIPLIVIFFCYGQLVFTVKEAAAQQQESATTQKAEKEV TRMVIIMVIAFLICWLPYAGVAFYIFTHQGSDFGPIFMTIPAFFAKTSAV YNPVIYIMMNKQFRNCMVTTLCCGKNPLGDDEASTTVSKTETSQVAPA*

  10. Logo for a) Splice acceptor b) Splice Donor c) Initiator Met

  11. AG/GT CAG/NT exon 1 intron 1 exon 2

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