DNA extraction – Lee Kerkhoff

• 2.3 ml 40% glucose
• 2.0 ml 0.5 M EDTA
• 2.5 ml 1M Tris Hcl
• 3 M sodium acetate
• Molecular grade glycogen

• Buffer I (Store at 4º C)

50 mM glucose
10 mM EDTA
25 mM TrisHCl pH 8.0

• Lysozyme solution (make fresh)

10 ml Buffer I	
40 mg Lysozyme (4mg/ml final)

• Place 100 mg soil or 0.22 μm supor filter in 50 μL Buffer I and suspend cells by vigorously pipetting up and down.
• Freeze-thaw sample in -80°C (liquid nitrogen?) and 37°C six to eight times
• Add:

200 μL Solution I
100 μL Lysozyme solution 
50 μL 0.5 M EDTA

• Place sample in roller aparatus (agitator) for 5-15 minutes at 37ÚC
• Add:

50   μL 10% SDS
800 μL Phenol:Chloroform:Isoamyl alcohol (24:1:1)

• Vortex 1-2 minutes to form emulsion
• Spin at 16 krpm (16k G) for three minutes
• Recover aqueous phase and repeat phenol extraction
• Precipitate DNA by adding:

50 μL 3 M sodium acetate 
100 μL 100% ethanol
1 μL glycogen (as carrier)

• Precipitate at -20°C for 15-30 minutes (30 min at -80°C better ?)
• Resuspend pellet in nuclease free water
• Use CsCl gradient to remove PCR inhibitor