(Birnboim/Doly)

• Put 2.0 ml of an O/N culture in an Eppendorf tube.
• Spin down for 1 minute at max rpm
• Resuspend the pellet in 200 ml TGE
• Add 400 ml fresh-made 0.2 M NaOH/1% SDS
• Mix well and incubate at RT for 5 minutes
• Add 300 ml 3M KAc pH 5.5
• Centrifuge for 10 minutes at max rpm.
• Put the supernatant in a clean vial (2ml Eppie)
• Add 500 ml isopropanol
• Spin down for 5 minutes at max rpm.
• Discard the supernatant.
• Dissolve the pellet in 300 ml H₂0
• Add 100 ml 10N NH₄Ac
• Leave the tube for 20 minutes on ice.
• Spin down for 10 minutes at 15000 rpm at 0 °C.
• Transfer the supernatant to a clean tube (careful, 1.5 ml eppie)
• Precipitate with 2 volumes 96% ethanol
• Spin down for 5 minutes at max rpm.
• Discard the supernatant and wash the pellet with 0.5 ml 70 % ethanol
• Spin down for 2 minutes and discard again the supernatant.
• Dry the pellet.
• Dissolve the pellet in 50 ml H₂0.

Reagents:

3M KAc pH 5.5

0.22 M NaOH

10 N NH₄Ac

10% SDS

TE: 10 mM Tris/HCl, 1mM EDTA, pH 8.0

TGE: 25 mM Tris/HCl, 50 mM Glucose, 10 mM EDTA