DAY 1

• Inoculate 5 ml LB broth with 50 ul P1 glycerol stock. Grow 5 hours.
• Inoculate 500 ml of LB containing 25 ug/ml kanamycin with 2-4mls of the fresh 5 hour P1 culture. Grow 16-18 hours.

DAY 2

• Spin cells at 5500 rpm in a 500 ml bottle in JA-10 rotor for 10 min.
• Discard supernatent and resuspend pellet in 60 ml of GTE buffer (solution 1).
• Add 0.3g of powdered lysozyme and incubate 30 min at R.temp.
• Add 120 ml alkaline-SDS (solution 2, prepared just prior to use).
• Mix gently and put on ice for 30 min.
• Add 40 ml KOAc (solution 3).
• Mix gently and put on ice for 10 min.
• Spin at 9000 rpm for 20 min in JA-10 rotor.
• Strain supernatent through a 2"X2" gauze pad into a 500 ml centrifuge bottle.
• Fill bottle with iso-propanol (at least 0.6 vol.). Store at -20 C overnight.

DAY 3

• Spin at 10,000 rpm for 5 min.
• Discard supernatent.
• Resuspend the pellet in 9.5 ml of TE-3 buffer (10mM Tris-Hcl 7.4, 1mM Edta) with gentle shaking for 15 min.
• Transfer to a 50 ml conical polypropylene centrifuge tube.
• Add 10.5 g of CsCl and mix gently to dissolve.
• Add 1.ml of ethidium bromide (10 mg/ml).
• Spin at 3000 rpm for 10 min in J-6 centrifuge.
• Transfer liquid to a 3" X 5/8" ultracentrifuge tube and seal.
• Spin 16 - 20 hours at 55,000 rpm. in 70.1 Ti rotor.

DAY 4

• Collect lower supercoiled band and transfer to a 15 ml conical centrifuge tube.
• Extract 5X 2 ml with water-saturated n-Butanol.
• Add 4ml water.
• Transfer to 50 ml.conical centrifuge tube.
• Fill tube to 45 ml with abs.EtOH and store at -20 C overnight.

DAY 5

• Spin at 3500 rpm for 30 min. in J-6 centrifuge.
• Wash pellet with 1 ml of 70% EtOH.
• Remove EtOH and air dry pellet.
• Resuspend pellet in 200 ul of TE-4.
• Incubation at 37 C aids in dissolvin g the DNA.
• Add 5 ul RNAse (500ng/ul) and incubate at 37 C for 60 minutes.
• Extract with phenol-chlorophorm.
• Ethanol precipitate.
• Dissolve pellet in 200 ul TE-4 (10mM Tris 7.4, 0.1mM edta)

Determine concentration by O.D.

YIELD:	5ug = poor	300-500ug = average
	Can get up to 1 microgram!

SOLUTION 1

SOLUTION 2
PREPARE JUST PRIOR TO USE

SOLUTION 3