Biotech Glossary | Bioinformatics | Lab Protocol | Notes | Malaysia University |
CONTRIBUTOR:
OVERVIEW:
PROCEDURE:
The Laboratory of Donald Rio at the University of California, Berkeley
This is a quick procedure to isolate plasmid DNA from transformed bacterial cells.
1. Pick a single transformed bacterial colony from a plate with a sterile toothpick.
2. Grow the bacteria in 1.5 ml of LB (with appropriate antibiotic) overnight with shaking at 37°C
3. Transfer the culture to a microfuge tube and centrifuge in a microcentrifuge at maximum speed for 5 min. Discard the supernatant.
4. Resuspend the cells in 300 μl of STET Buffer. Vortex the tube to disperse any cell clumps.
5. Add 5 μl of Lysozyme Solution and mix well.
6. Place the tubes in a boiling water bath for 40 sec.
7. Centrifuge the tube immediately at full speed in a microcentrifuge for 10 min at room temperature.
8. Remove the viscous, opaque pellet of bacterial cell wall debris and chromosomal DNA from the microcentrifuge tube with a new toothpick.
9. Add 1 μl of RNase A solution.
10. Incubate for 10 min at room temperature for samples that are to be used for DNA digests. Incubate for 45 min at room temperature for samples that will be used for double-stranded DNA sequencing.
11. Add 150 μl of Phenol and vortex the tube for 10 sec (CAUTION! see Hint #1).
12. Centrifuge at room temperature in a microcentrifuge at full speed for 1 min and collect the upper (aqueous) phase.
13. Add 600 μl of Chloroform to the aqueous phase and vortex the tube.
14. Centrifuge at room temperature in a microcentrifuge at full speed for 1 min and collect the aqueous (upper) phase.
15. Add 400 μl of -20°C Isopropanol and centrifuge for 5 min at full speed in a microcentrifuge.
16. Discard the supernatant and drain the liquid from the tube as completely as possible. Add 500 μl of 70% Ethanol to the tube and resuspend the pellet.
17. Centrifuge for 5 min in a microcentrifuge at full speed.
18. Discard the supernatant and drain the liquid as much as possible and dry the pellet.
19. Dissolve the DNA in 25 μl of TE Buffer if it is to be used for a restriction digest. Use 5 μl for one restriction enzyme digest with 5 Units of Restriction Enzyme (see Hint #2).
OR
20. Resuspend the DNA pellet in 100 μl of 2.5 M Ammonium Acetate if it is to be used for sequencing. Then add 250 μl of 100% Ethanol and vortex the tube.
21. Freeze the sample in a Dry Ice/Ethanol bath for 10 min.
22. Centrifuge at full speed in a microcentrifuge at 4°C for 10 min.
23. Discard the supernatant and resuspend the pellet in 100 μl of 0.3 M Sodium Acetate and add 250 μl of 100% Ethanol and vortex the tube.
24. Freeze the sample in a Dry Ice/Ethanol bath for 10 min.
25. Centrifuge at full speed in a microcentrifuge at 4°C for 10 min.
26. Remove the supernatant and dry the DNA in a vacuum centrifuge.
27. Dissolve the DNA in 20 μl of ddH2O and store at -20°C until sequencing (see Protocol on Sequencing DNA).
SOLUTION:
2.5 M Ammonium Acetate
TE Buffer
10 mM Tris
pH 8.0
1 mM EDTA
70% (v/v) Ethanol
Chloroform
24:1 Chloroform:Isoamyl Alcohol (CAUTION! see Hint #1)
RNase A Solution
Boil for 10 min and cool to room temperature
10 mg/ml RNase A (Worthington)
Store at -20°C
Lysozyme Solution
20 mg/ml Lysozyme
Store at -20°C
STET Buffer
50 mM EDTA
50 mM Tris, pH 8.0
8% (v/v) Sucrose
5% (v/v) Triton X-100
LB
5 g/liter NaCl
5 g/liter Yeast Extract
10 g/liter Tryptone
1 ml/liter 1.0 M NaOH
Autoclave
0.3 M Sodium Acetate
REAGENTS AND CHEMICALS:
Sodium Acetate
Ethanol
Triton X-100
Sucrose
RNase A
Sodium Hydroxide
Isopropanol
Isoamyl Alcohol
Chloroform
Lysozyme
Phenol
Dry Ice
Tris
Ammonium Acetate
Yeast Extract
Tryptone
Restriction Enzyme
EDTA
Sodium Chloride
PROTOCOL HINTS:
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
2. These volumes are usually sufficient to visualize the DNA in an agarose gel after digestion. Skip Steps #20 to #27.