Toronto/Lab Protocols/Miniprep

From 2007.igem.org

Miniprep Procedure:

Small-Scale Plasmid Preparation

1. Put 1.5 mL O/N into a 1.5 mL Eppendorf tube.
2. Centrifuge for 1 minute.
3. Aspirate the supernatant.
4. In order to increase the concentration of DNA, add the rest of the o/n into the same tube and centrifuge for 1 minute again.
5. Aspirate the supernatant and resuspend cells with 250 μL Resuspension Solution (red). Vortex until pellet dissolves.
6. Add 250 μL Lysis Solution and mix with pipette. (shake with hand)
7. Sit for 3-5 minutes.
8. Add 350 μL Neutralization Solution (blue) and invert tube with hand 4-6 times.
9. Centrifuge for 5 minutes.
10. Pour supernatant in a column tube.
11. Centrifuge for 1 minute and throw away supernatant.
12. Add 500 μL Wash Solution.
13. Centrifuge for 1 minute and throw away supernatant.
14. Add 500 μL Wash Solution.
15. Centrifuge for 1 minute and throw away supernatant.
16. Put blue column in a fresh centrifuge tube. (make sure to label)
17. Add 50 μL Elution Buffer.
18. Incubate for 2 minutes at room temperature, then centrifuge for 2 minutes.
19. Throw away blue column.
20. Place plasmid into the freezer.

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