Plasmid isolation

• transfer 2 x 1.5 ml of overnight culture into an eppendorf tube. Pellet cells for 30 sec at 14,000 RPM .
• remove all liquid.
• resuspend cells in 200 ul of solution I.
• add 200 ul of solution III, mix by 1-2 gentle rotation.
• add 200 ul of ice-cold solution III, mix well .
• Centrifuge 14,000 rpm for 5 min in cold room.

• transfer supernatant to fresh tube.
• add 1 ml of ethanol, mix, centrifuge14,000 rpm for 5 min at RT.
• wash the pellet with 1 ml of 80% ethanol and Spin Vac dry.
• dissolve the pellet in 50 ul of water and add 50 ul of 7.5 M NH4OAc, mix, incubate on ice for 5 min, centrifuge14,000 rpm for 5 min in the cold room.
• transfer supernatant to fresh tube and add 50 ul of isopropanol, mix, centrifuge 14,000 rpm at RT for 5 min.
• wash pellet with 80% ethanol and Spin Vac dry.
• dissolve pellet in 20 ul of water.
• use 5 ul for sequencing and 1 ul for restriction analysis.