Biotech Glossary | Bioinformatics | Lab Protocol | Notes | Malaysia University |
CONTRIBUTOR:
OVERVIEW:
PROCEDURE:
The Laboratory of Jasper Rine at the University of California, Berkeley
This is a quick procedure to isolate plasmid DNA from transformed bacterial cells.
1. Grow a 3 ml overnight culture of transformed E coli to saturation.
2. Add 1.5 ml of the E coli culture to a microcentrifuge tube and centrifuge it in a microcentrifuge for 2 min at 14,000 rpm.
3. Aspirate the supernatant.
4. Resuspend the pellet in 300 μl of STET (See Hint #2).
5. Add 20 μl of Lysozyme Solution to the tube.
6. Mix and incubate at room temperature for 15 sec to 10 min (incubate longer for smaller pellets).
7. Place in a boiling water bath for 2 min (with caps of tubes open).
8. Incubate the tube on ice for 5 min.
9. Centrifuge in a microcentrifuge for 5 min at 14,000 rpm.
10. Scoop out viscous opaque bacterial cell wall and chromosomal material with a sterile toothpick.
11. Add to the supernatant an equal volume of 75% Isopropanol, 2.5 M Ammonium Acetate Solution.
12. Mix and centrifuge in a microcentrifuge for 5 min at 14,000 rpm. Aspirate the supernatant.
13. Wash the pellet with 70% Ethanol and centrifuge in a microcentrifuge for 5 min at 14,000 rpm.
14. Resuspend the pellet in 200 μl of 0.3 M NaCl.
15. Add 10 μg of RNase A from the RNase A Stock, mix, and incubate the tube at room temperature for 30 min.
16. Add an equal volume of Phenol:Chloroform:Isoamyl Alcohol to each tube and mix vigorously for a few seconds by vortexing.
17. Centrifuge the samples at maximum speed in a microcentrifuge for 5 min to separate the aqueous and organic phases. Remove the aqueous phase to new tubes.
18. Add an equal volume of Chloroform:Isoamyl Alcohol to the aqueous phase and mix vigorously for a few seconds by vortexing.
19. Centrifuge the samples at maximum speed in a microcentrifuge for 5 min to separate the aqueous and organic phases.
20. Transfer the aqueous phase to a new tube and add 2.5 volumes of 100% Ethanol and mix.
21. Incubate at -20°C for 30 min.
22. Centrifuge the samples at maximum speed in a microcentrifuge for 5 min.
23. Wash the pellet with 70% Ethanol and centrifuge in a microcentrifuge for 5 min at 14,000 rpm.
24. Lyophilize the pellet and resuspend it in 10 to 20 μl of ddH2O.
SOLUTION:
Chloroform:Isoamyl Alcohol
24:1 Chloroform:Isoamyl Alcohol (CAUTION! see Hint #1)
Phenol:Chloroform:Isoamyl Alcohol
Store at 4°C in a dark glass container
25:24:1 Phenol:Chloroform:Isoamyl Alcohol (CAUTION! see Hint #1)
Lysozyme Solution
50 mM Tris, pH 8.0
10 mg/ml Lysozyme
STET
50 mM EDTA
50 mM Tris, pH 8.0
8% (w/v) Sucrose
5% (v/v) Triton X-100
RNase A Stock
10 mg/ml RNase A
70% (v/v) Ethanol
0.3 M NaCl
75% Isopropanol, 2.5 M Ammonium Acetate Solution
75% (v/v) Isopropanol
2.5 M Ammonium Acetate
REAGENTS AND CHEMICALS:
Triton X-100
Ethanol
Sodium Chloride
EDTA
Ammonium Acetate
Tris
Isopropanol
Lysozyme
Sucrose
RNase A
Chloroform
Phenol
PROTOCOL HINTS:
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
2. At this point, up to 3 pellets can be pooled in the same 300 μl of STET.