Large-scale Plasmid Isolation
From CCGB
Large-scale Plasmid Isolation
-- by Hao Wang
1. The DH5α harboring a plasmid was grown in the presence of 100μg/ml ampicillin until it reaches the late log phase.
2. Pellet cells by centrifugation @ 5860g for 6 min. repeated centrifugations may be performed.
3. Add 15ml of Cell Resuspension Solution with lysozyme. Complete resuspension is critical for optimal yields.
4. Add 35 ml of Cell Lysis Solution and mix gently, but thoroughly, by stirring or inverting. Do not vortex. Cell lysis is complete when the solution becomes clear and viscous (up to 20 min, keep on ice).
5. Add 15 ml of Neutralization Solution and immediately mix by inverting the centrifuge bottle several times (up to 30 min, keep on ice).
6. Centrifuge @ 12100g for 15 min @ 4°C.
7. Carefully decant the cleaned supernatant to a new centrifuge bottle, avoiding the white precipitate. Alternatively, transfer the cleared supernatant by filtering it through whatman#1 filter paper into the new centrifuge bottle.
8. Add 0.6 volumes of Isopropanol and mix by inversion. Keep for 15 min @ room temperature.
9. Centrifuge @ 12100g for 15 min @ 4°C.
10. Discard the supernatant, and resuspend the DNA pellet in 9ml of TE (pH8.0).
11. Add 10.12g CsCl to each preparation.
12. Transfer to the Beckman centrifuge tubes (plastic, frosted). Use glass Pasteur pipets.
13. Put into balance, add Ethidium bromide (10mg/ml in water) to the top. Use syringe.
14. Seal the top and check the balance again.
15. Centrifuge @ 45000rpm for a minimum of 36 hours in the Ti70.1 rotor.
16. After centrifugation, DNA bands separated.
17. Collect the lower band of closed-circular plasmid DNA.
a. Insert 2×21-gauge hypodermic needle into the top of the tube to allow air to enter.
b. Insert 18-gauge hypodermic needle (beveled side up) into the tube (vevelled side of the needle is positioned just below the band of closed circular plasmid DNA).
c. Collect the viscous DNA into a disposable tube.
d. Seal the tube with a piece of Scotch tape.
18. Bring volume to 3ml with TE
19. Add equal volume of isoamyl alcohol.
20. Mix the two phases by vortexing.
21. Centrifuge the mixture @ 1500rpm for 3 min @ room temprature in a bench centrifuge.
22. Using a Pasteur pipet transfer the lower aqueous phase to a clean corex tube.
23. Repeat the extraction 4-6 times until the ink color disappears from both the aqueous phase and organic phase.
24. Dilute sample in TE up to 6 ml.
25. Add 3 ml 7.5 M NH4Oac.
26. Add 2 volumes of 100% Ethanol. Store @ -20°C for 1 hr. Spin for 15 min @ 12100f.
27. Wash with 70% Ethanol. Spin. Dry at the bench.
28. Resuspend DNA in 500-1000μl of sterile ddH2O in Eppendorf tube.
Cell Resuspension Buffer with Lysozyme
50mM glucose/10 mM EDTA/25 mM Tris-HCL, pH8.0; 4 mg lysozyme/ml
Cell Lysis Buffer
0.2M NaOH/1% SDS
Neutralization Solution
2.5 M NH4Oac without pH adjustment
