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exercise 10 - CHAPTER 10 LAB 10 Alkaline Lysis mini-prep to...

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CHAPTER 10/ LAB 10 Alkaline Lysis mini-prep to isolate plasmid from bacterial cells DNA replication PCR to amplify large amounts of DNA in a few hours PLASMID o Small, circular DNA in prokaryotes o Carries non-essential genes antibodies, metabolizing unusual food sources, producing toxin o Transferred by Transformation or Conjugation ( mating process) Selection: selective media o Containing an antibiotic o Containing or lacking a specific required nutrient Stringent control: replicates when chromosome does; only 1 copy Relaxed control : uncoupled with chromosomes hundreds of copies 3 states of plasmids: o Supercoiled : favorable state o Coiled : one coil from supercoiled is broken (nicked backbone); open circular o Linear : Backbone of both strands of double helix are broken Goal of Plasmid Extraction : o Separate plasmid DNA from chromosomal DNA & protein o ALKALINE LYSIS PROCEDURE: 1. Grow bacteria overnight in Luria Browth and centrifuge 2. Resuspend in STE buffer ( NaCl, Tris buffer, EDTA at pH 8) 3. Centrifuge, keep pellet cells, PELLET in GTE BUFFER (Tris, glucose, EDTA) 4. pH 8 5. ADD SDS detergent(contains NaOH) solution (pH = 12) Completely denatures chromosomal DNA Partially denaturing plasmid DNA 6. Ice for 5 minutes
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7. Add Potassium Acetate (pH 5.5) to precipitate cell debris (proteins, chromosomal DNA) 8. Vortex and chill for 5 min. 9. Put in microcentrifuge at 4 o C for 5 minutes 10.Discard pellet, keep supernatant 11.Centrifuge again, with 95% ethanol – 3 minutes at 80% full speed 12.Pour supernatant into 25 mL beaker, KEEP PELLETS 13.Transfer 1mL of 70% ethanol 14.Centrifuge again,
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