Biotech Glossary | Bioinformatics | Lab Protocol | Notes | Malaysia University |
OVERVIEW:
Large scale highly purified plasmid DNA preparation from bacterial cells.
PROCEDURE:
1. Centrifuge to pellet cells (approximately 1,500 X g), discard the supernatant and resuspend the cells in 5 ml of TE Suspension Solution.
2. Add 10 ml of freshly prepared NaOH/SDS Solution and mix thoroughly by inversion.
3. Incubate at room temperature for 2 to 3 min (do not exceed 3 min).
4. Add 7.5 ml of Acetate Solution and vortex.
5. Centrifuge for 10 min at approximately 5,900 X g.
6. Save the supernatant and filter the supernatant through cheese cloth.
7. To the filtered supernatant add 11 ml of 100% Isopropanol and mix well by inversion.
8. Centrifuge for 10 min at approximately 5,900 X g.
9. Discard the supernatant and resuspend the DNA pellet in 3 ml of TE Buffer.
10. Add 100 μl of Ethidium Bromide and 4.4 g CsCl.
11. Bring the final volume of the solution to 4.3 ml using TE buffer.
12. Centrifuge in an ultracentrifuge at 20°C for at least 6 hours (cushion may take as long as 12 to 18 hours) at 267,000 X g (55,000 rpm using a VTi65 rotor).
13. Illuminate the Ethidium Bromide band by shinning a hand-held UV lamp on the tube.
14. Remove the Ethidium Bromide band and place in a new ultracentrifuge tube.
15. Bring the final volume of the tube to 10 ml using Cesium/TE Solution.
16. Centrifuge in an ultracentrifuge as in Step #12 and remove the Ethidium Bromide band as in Steps #13 and #14. Save the Ethidium Bromide band solution and add this to a new tube.
17. To the Ethidium Bromide band solution add 3 to 4 volumes of 1-Butanol Solution.
18. Mix well by inversion and centrifuge to separate the phases.
19. Save the aqueous phase and check under UV lamp for Ethidium Bromide illumination. Repeat the 1-Butanol wash until there is no Ethidium Bromide illumination.
20. To the aqueous phase that is free of Ethidium Bromide, add an equal volume of TE Buffer and 5 volumes of room temperature 100% Ethanol.
21. Centrifuge immediately to precipitate the DNA.
22. Decant the supernatant and allow the DNA pellet to air dry for a couple of minutes.
23. Resuspend the DNA pellet in 400 μl of TE Buffer.
24. Precipitate the DNA by adding 1 ml of 100% Ethanol and 35 μl of 5 M NaCl.
25. Centrifuge in a microcentrifuge to precipitate DNA.
26. Decant the supernatant and allow DNA pellet to air dry for a couple of minutes.
27. Resuspend the DNA pellet in 400 μl of TE Buffer.
SOLUTION:
Ethidium Bromide
in TE buffer
10 mg/ml Ethidium Bromide 
TE Buffer
10 mM Tris
pH 8.0
1 mM EDTA
Acetate Solution
11.5 ml of Glacial Acetic Acid
28.5 ml of ddH2O
60 ml of 5 M Potassium Acetate
NaOH/SDS Solution
1% (w/v) SDS
Prepare just before use
0.2 M NaOH
5 M NaCl
TE Suspension Solution
10 mM EDTA
25 mM Tris
pH 8.0
1-Butanol Solution
Mix well
Use the 1-Butanol and discard the ddH2O
10 ml ddH2O
Saturate overnight at 4°C
100 ml 1-Butanol
Cesium/TE Solution
10 ml TE Buffer
10 g Cesium Chloride (CsCl)
REAGENTS AND CHEMICALS:
SDS
Potassium Acetate
Tris
Ethanol
Ethidium Bromide
EDTA
Glacial Acetic Acid
Cesium Chloride
Isopropanol
Cheesecloth
1-Butanol
Sodium Chloride
Sodium Hydroxide