1. Laboratory: Unit 3: prepare genomic DNA (53-54) Lecture: Genomic DNA purification In-Class Writing: discuss JBC 266: 3811-14 (1991) (page 155) Hand In: flow chart 3; lab report 2 Read: AEM 63: 2647-2653, 1997 (minus abstract) (see page 68) Due Next Class: abstract for AEM 63: 2647-53

2. Use one isolate. Pick a dense culture. Transfer 1 ml culture to sterile vial + 0.2 ml DMSO; shake to mix. Label with your seat number. Wrap tape around vial so ends overlap. Place in freezer box. Place remaining culture in 1.5-ml tube; purify genomic DNA (section D).

3. Purify Genomic DNA 1. Transfer 1.5 ml culture to centrifuge tube. 2. Centrifuge maximum speed 1 minute. Discard supernatant (autoclave). 3. Suspend cells in 450  l of Tris + EDTA; disperse pellet completely. EDTA chelates Mg ++  inhibits nucleases & helps disrupt cells.

4. 4. Add 20  l lysozyme, 20 min, 37 o C. Lysozyme cleaves peptidoglycan cell wall. 5. Add 10  l proteinase K, 20 min, 50 o C. Proteinase K cleaves cell wall proteins & nucleases.

5. 6. Add 20  l 25% SDS, 10 min, 68 o C. SDS = ionic detergent; disrupts membrane lipids & denatures proteins. 7. Add 57  l 5 M NaCl; vortex 1 minute. DNA & RNA precipitate in high salt/ethanol.

9. 8. Incubate 5 min, 68 o C; vortex 1 minute. Shears DNA; reduces viscosity. 9. Add 0.5 ml phenol:chloroform:isoamyl alcohol (25:24:1) equilibrated with 1 M Tris. Mix well. Avoid contact with organic solvents; work in fume hood. Lab coats, gloves, goggles, closed shoes required. If phenol contacts skin, wash with water & seek medical attention. 10. Centrifuge maximum speed, 5 minutes.

10. 11. Transfer aqueous (top) phase to 1.5-ml tube with 0.5 ml chloroform:isoamyl alcohol (24:1). Withdraw top phase; leave cell debris at interphase. DNA attached to membrane will tug at interphase. Discard organic solvents in organic waste. 12. Mix well; centrifuge maximum speed, 2 min.

11. 13. Transfer aqueous (top) phase to 1.5-ml tube. Leave cell debris at interphase. Discard organic solvents in organic waste. 14. Add 1 ml ethanol (ice cold), mix thoroughly; hold 15 min, 0 o C or 4 o C. 15. Centrifuge maximum speed, 5 min; orient tube in rotor so pellet forms at known location. Discard supernatant solution.

12. 16. Wash DNA/RNA pellet with 0.5 ml 70% ethanol. Let stand 1 minute; do not disturb pellet. 17. Centrifuge maximum speed, 2 minutes. Discard supernatant solution carefully; pellet will not adhere tightly to tube.

13. 18. Centrifuge 10 seconds. Remove ethanol with pipet tip. Air dry pellet. 19. Dissolve DNA/RNA pellet in 50  l DNA buffer. Freeze at -20 o C.