Small scale isolation of plasmid DNA

• prepare solution II (1,5 ml for 10 präps)
  2x600 µl water
  150 µl 10% SDS
  150 µl 2M NaOH
• transfer 1,5 ml of an overnight culture in LB into tube
• spin 1min
• aspirate supernatant completly
• suspend bacteria in 100 µl of solution 1)
• add 200 µl of solution II,
  incubate for 3 to 5 min, the suspension should clarify (bacteria are lysed)
• add 150 ml of solution III,
  mix briefly, but vigorously
• incubate for 5 min on ice,
  spin 5 min (4°C or RT)
• transfer supernatant (400 µl) to new tube,
  make sure not to take anything of the white precipitate
• transfer supernatant (400 µl) to a new (labelled) vial with 280 µl 2-propanol,
  mix, spin 10 min (4°C) (alternatively precipitate with 1000 µl ethanol, which leads to a more visible pellet than preciptiation by 2-propanol
• carefully aspirate supernatant,
  wash with 70% ethanol
• completely aspirate supernatant,
  dry for 1 min in speed-vac
• dissolve in 50 µl TE,
  use 5 µl for digest (add RNase A)

• after precipitation with solution III, transfer supernatant (400 µl) to 400 µl phenol:chloroform.i-amylalkohol, shake vigorously for about 1 min, spin 5 min
• transfer aqueous phase (upper layer) to new tube with 400 µl chloroform, mix vigorously, spin 3 min
• transfer supernatant to 270 µl 2-propanol, mix, spin 10 min
• aspirate carefully supernatant, wash with 70% ethanol
• aspirate supernatant completely, dry for 1 min in speed-vac
• dissolve in 50 µl TE, use 5 µl for digest