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Mini-prep Protocol
From 2012.igem.org
(Created page with "1. Different cultures (each one with a different construction), which are growing in a selective media (LB + Ampicillin), get centrifuged at 4500g 5 min. 2. Supernatant is remov...") |
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9. Mix it inverting the tube 4-6 times. Incubate in ice for 15-30 min. | 9. Mix it inverting the tube 4-6 times. Incubate in ice for 15-30 min. | ||
| - | ''Now if it | + | ''Now if it was necessary, the process could stop here keeping the eppendorf tube in ice.'' |
10. Centrifuge 10’ (max. rpm) in order to pellet cell debris and chromosomal DNA. | 10. Centrifuge 10’ (max. rpm) in order to pellet cell debris and chromosomal DNA. | ||
Latest revision as of 09:32, 21 August 2012
1. Different cultures (each one with a different construction), which are growing in a selective media (LB + Ampicillin), get centrifuged at 4500g 5 min.
2. Supernatant is removed.
3. The cells can be washed (x2) with a saline solution (PBS) in order to remove impurities.
4. The pellet is resuspended in 250 μL of Resuspension Solution (RNase A added to it previously. This solution is kept at 4ºC). Important: resuspend it completely.
5. Transfer the suspension to an eppendorf tube.
6. Add 250 μL of Lysis Solution.
7. Mix it inverting the tube 4-6 times (DO NOT VORTEX!) until solution gets viscous and slightly clear. Important: Do not incubate more than 5 min.
8. Add 350 μL of Neutralization Solution.
9. Mix it inverting the tube 4-6 times. Incubate in ice for 15-30 min.
Now if it was necessary, the process could stop here keeping the eppendorf tube in ice.
10. Centrifuge 10’ (max. rpm) in order to pellet cell debris and chromosomal DNA.
11. Transfer the supernatant (≈ 800 μL) to the spin column (pipetting to avoid carrying impurities). Important: DO NOT TRANSFERING THE PRECIPITATE!
12. Centrifuge 1’.
13. Flow-though liquid is removed.
14. Add 500 μL of Wash Solution (Solution stock has to be perfectly closed, it contains ethanol!).
15. Centrifuge ≈ 1’.
16. Flow-though liquid is removed.
17. 14, 15, 16 steps are repeated.
18. Centrifuge 1’ in order to eliminate residual Wash Solution.
19. The spin column is transferred into an eppendorf tube (the collection tube is eliminated).
20. Add 50 μL of Elution Buffer to the center of spin column membrane and let it 5’ getting soaked (it increases the efficiency of process). Important: DO NOT CONTACT THE COLUMN MEMBRANE WITH THE PIPETTE TIP!
21. Centrifuge ≈ 2’.
22. To increase the efficiency (≈ 20%) we can get the flow-though liquid and repeat the steps previously described (20 and 21).
23. The column is discarded and the solution which contains the purified plasmid can be stored in cold.
