MINIPREP

Materials

• Resuspension buffer
• Lysis buffer
• Neutralization buffer
• Isopropanol
• 70% Etanol
• dH2O
• Ice (in ice bucket/container)

Procedure

1. Centrifuge for 5 minutes at 5000 rpm 1,5 ml of bacteria culture.
2. Take the pellet and add 250 ul of resuspension buffer and pipet up and down a few times.(make sure that all is properly mixed)
3. Add 250 ul of Lysis buffer and invert the tubes 5 times to mix. Let rest 5 minutes. The mix must turn transparent.
4. Add 250 ul of cold Neutralization buffer and invert the tubes 5 times to mix. The mix must turn turbid.
5. Centrifuge at 13.000 rpm for 15 minutes.
6. Transfer the supernatant to a new clean eppendorf tube.
7. Add isopropanol (the same volume that is in the tube). Mix many times and put it in ice for 20 minutes.
8. Centrifuge at 13.000 rpm for 20 minutes.
9. Eliminate the supernatant (be carefull that the pellet does not go with it)
10. Add 1 ml of etanol 70% and use a vortex to ensure the pellet mixes with the etanol.
11. Centrifuge at 13.000 rpm for 5 minutes.
12. Eliminate all the supernatant with a pipette (be carefull that the pellet does not go with it)
13. Let the tube open until it is dried. The pellet must turn from white to transparent.
14. Add 20 ul of dH2O and freeze.