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prepare foreign (target) DNA prepare vector (host) recombine target and vector DNA introduce rDNA to host screen for DNA

gDNA (fragments). cDNA (copy of RNA). Preparing gDNA restriction enzymes ‘random nuclease’  size fractionate. Recombinant DNA. prepare foreign (target) DNA prepare vector (host) recombine target and vector DNA introduce rDNA to host screen for DNA of interest . gDNA vs cDNA.

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prepare foreign (target) DNA prepare vector (host) recombine target and vector DNA introduce rDNA to host screen for DNA

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  1. gDNA (fragments) cDNA (copy of RNA) • Preparing gDNA • restriction enzymes • ‘random nuclease’ •  size fractionate Recombinant DNA • prepare foreign (target) DNA • prepare vector (host) • recombine target and vector DNA • introduce rDNA to host • screen for DNA of interest

  2. gDNA vs cDNA • level of expression • differential gene expression • accessibility • introns • complicates gDNA analysis • can preclude expression • ease of preparation • cDNA more work

  3. Preparation of cDNA • 1) Isolate mRNA • 2) Synthesize DNA-RNA hybrid • reverse transcriptase • oligo-dT primer • random priming • 3) Synthesize 2nd DNA strand • 4) Add termini  RNA dependent DNA polymerase

  4. 1) Isolate mRNA • 2) Synthesize DNA-RNA hybrid • 3) Synthesize 2nd DNA strand • self-priming • replacement synthesis • primed synthesis • 4) Add termini

  5. 1) Isolate mRNA • 2) Synthesize DNA-RNA hybrid • 3) Synthesize 2nd DNA strand • 4) Add termini • i.e., linkers CGGAATTCCG GCCTTAAGGC Eco RI

  6. Recombinant DNA Vectors • autonomously-replicating DNA used to ‘carry’ and amplify foreign DNA within host cell • eg: plasmids, phage/viruses, or combinations • Plasmids • extra-chromosomal elements • 1-200 kb size range • transmitted during conjugation • antibiotic resistance • low copy number vs high copy number

  7. Multiple Cloning Site: |SacI||ScII||XbaI||SpeI||BamH||SmaI||PstI||EcRI||EcRV||HIII||ClaI||SalI||XhoI||KpnI| GAGCTCCACCGCGGTGGCGGCCGCTCTAGAACTAGTGGATCCCCCGGGCTGCAGGAATTCGATATCAAGCTTATCGATACCGTCGACCTCGAGGGGGGGCCCGGTACC CTCGAGGTGGCGCCACCGCCGGCGAGATCTTGATCACCTAGGGGGCCCGACGTCCTTAAGCTATAGTTCGAATAGCTATGGCAGCTGGAGCTCCCCCCCGGGCCATGG Useful Plasmid Features • Relaxed Replication • Selectable Markers • Streamlined • Polylinker or MCS • Identification of Recombinants • most derived from pUC or pBR322

  8. Generic rDNA Protocol • prepare foreign DNA • prepare vector • ligate foreign DNA and vector • introduce vector into host • screen for rDNA of interest

  9. Ligation Reaction • mix foreign and vector DNA in presence of DNA ligase • optimal ratios of vector to insert generally 1.5-2:1 • intermolecular base-pairing can occur between compatible overhangs

  10. DNA Ligase • catalyzes the formation of phosphodiester bond between 5’-PO4 and 3’-OH • i.e., joins DNA fragments • typically carried out at lower temperatures (8-16o) for extended periods

  11. Intramolecular vs. Intermolecular

  12. Removal of 5’-PO4 Prevents Vector Self Ligation

  13. prepare foreign DNA • prepare vector • ligate target and vector • introduce rDNA to host • heat shock + Ca2+ • electroporation • select for transformants with antibiotic • screen for rDNA of interest

  14. Colony Lift • Sources of Probes • cloned genes • synthetic oligonucleotides • PCR products

  15. Identifying Recombinants • based on interruption of a gene • eg., lacZ gene = b-galactosidase • intact b-galactosidase produces blue color in presence of X-gal • -complementation or blue-white screening

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