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Team:Valencia Biocampus/Protocols
From 2012.igem.org
Protocols
Heat Shock
1) Take competent E.coli cells from –80°C freezer.
2) Turn on water bath to 42°C.
3) Put 100 ul of competent cells in an Eppendorf tube.
4) Keep tubes on ice.
5) Add 50 ng of circular DNA into E.coli cells. Incubate on ice for 10 minutes to thaw competent cells.
6) Put tube(s) with DNA and E.coli into water bath at 42°C for 45 seconds.
7) Put tubes back on ice for 2 minutes to reduce damage to the E.coli cells.
8) Add 1 ml of LB (with no antibiotic added). Incubate tubes for 1 hour at 37°C.
9) Spread about 100 ul of the resulting culture on LB plates (with Ampicillin added). Grow overnight.
10) Pick colonies about 12-16 hours later.
Mini-prep
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.
