• Pellet microcarriers at 500g for 10 minutes at 4°C

• Resuspend cells in sterile lysis buffer, 4.75ml per 106 cells

400mM NaCl
10mM Tris pH 8.0
2mM EDTA pH 8.0

• Add SDS to 0.5% and gently mix until viscosity is apparent

• Add proteinase K to 200 µg/ml and incubate for 2 to 4 hours at 37°C, swirling periodically

• Gently extract the DNA three times:
1. with 1 volume saturated phenol
2. with 0.5 volume saturated phenol and 0.5 volume chloroform
3. with 1 volume chloroform

• Add RNAse A to 3.3µg/ml and incubate at 37°C for 30 minutes

• Dialyze extensively against TE at 4°C

• Precipitate DNA with 1/10 volume 3M Sodium Acetate and 2 volumes of ethanol

• Centrifuge DNA at 15000g or more for 1 hour

• Wash DNA pellet with 75% ethanol, air dry

• Resuspend in TE and measure concentration with spectrophotometer