Team:XJTLU-CHINA/Protocols Plasmid DNA isolation
Plasmid DNA isolation from Lactococcus lactis
Materials:
Procedure:
- Use 5 ml full grown culture
- Spin down 10 min 3000 x g or more (up to 6000 x g)
- Re-suspend pellet in 250 μl THMS-buffer + 2 mg/ml lysozyme in Eppendorf cup
- Incubate 10 min at 37 °C
- Add 500 μl 0.2 N NaOH + 1% SDS, shake carefully (no vortex)
- Incubate 5 min on ice
- Add 375 μl ice-cold 3 M potassium acetate pH 5.5, shake carefully
- Incubate 5 min on ice
- Spin 5 min in Eppendorf centrifuge (Maximum Rotation Speed)
- Take out supernatant and add to 2 ml new Eppendorf cup
- Add 0.7-1 ml 2-propanol
- Incubate 5-10 min at room temperature
- Spin 10 min in Eppendorf centrifuge (Maximum Rotation Speed)
- Wash pellet carefully with 70% ethanol
- Dry the pellet (vacuum)
- Dissolve the pellet in 50 μl TE or sterile water
(For our project, the plasmid we used is pNZ8148, which is chloramphenicol-resistant)
Applications in our project: pNZ8148 extraction
Collaborators and Supporters
