Plasmid DNA isolation from Lactococcus lactis

Materials:

Procedure:

1. Use 5 ml full grown culture
2. Spin down 10 min 3000 x g or more (up to 6000 x g)
3. Re-suspend pellet in 250 μl THMS-buffer + 2 mg/ml lysozyme in Eppendorf cup
4. Incubate 10 min at 37 °C
5. Add 500 μl 0.2 N NaOH + 1% SDS, shake carefully (no vortex)
6. Incubate 5 min on ice
7. Add 375 μl ice-cold 3 M potassium acetate pH 5.5, shake carefully
8. Incubate 5 min on ice
9. Spin 5 min in Eppendorf centrifuge (Maximum Rotation Speed)
10. Take out supernatant and add to 2 ml new Eppendorf cup
11. Add 0.7-1 ml 2-propanol
12. Incubate 5-10 min at room temperature
13. Spin 10 min in Eppendorf centrifuge (Maximum Rotation Speed)
14. Wash pellet carefully with 70% ethanol
15. Dry the pellet (vacuum)
16. 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