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

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

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,