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The Laboratory of Giovanna Ferro-Luzzi Ames at the University of California, Berkeley
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1. Add 1.5 ml of an overnight LB culture of bacteria into a microcentrifuge tube and centrifuge at maximum speed in a microcentrifuge for 1 min. Aspirate and discard the supernatant. Dry the pellet over a tissue if necessary.
2. Add 350 μl STET and vortex the tube to resuspend the cells. Add 25 μl freshly dissolved lysozyme solution and mix well. When all the samples are prepared, boil them for 40 sec.
3. Immediately centrifuge for 10 min at full speed in a microcentrifuge.
4. Remove the pellet with a toothpick. Add 40 μl of 2.5 M Sodium Acetate and 420 μl Isopropanol. Mix well by vortexing and then freeze in a dry ice/Ethanol bath for 15 min.
5. Centrifuge for 15 min at full speed in a microcentrifuge at 4°C and discard the supernatant. Allow the pellet to air dry. Dissolve the pellet in an appropriate buffer for digestion by restriction endonucleases, or in 50 μl TE Buffer and add RNase at 50 ug/ml and incubate at 37°C for 10 min.
6. Add 40 μl of 2.5 M Sodium Acetate and 420 μl Isopropanol. Mix well by vortexing and then freeze in a dry ice/Ethanol bath for 15 min. Centrifuge for 15 min at full speed in a microcentrifuge at 4°C and discard the supernatant. Allow the pellet to air dry and resuspend in 50 μl TE buffer.
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| TE Buffer |
| 10 mM Tris
pH 8.0
1 mM EDTA
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| 2.5 M Sodium Acetate |
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| STET |
| 50 mM Tris-HCl, pH 8.0
50 mM EDTA
8 % (w/v) Sucrose
0.5 % (v/v) Triton X-100
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| Lysozyme Solution |
| Prepare fresh just before use
10 mg/ml Lysozymes in 10 mM Tris, pH 8.0
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| LB Broth |
| 2 % (w/v) Tryptone
10 mM NaCl
0.5 % (w/v) Yeast Extract
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Tris-HCl
Triton X-100
Ethanol
Sodium Chloride
Dry Ice
Yeast Extract
EDTA
Tryptone
Isopropanol
Lysozyme
Sucrose
Sodium Acetate
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No hints are associated with this bioProtocol
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1. Holmes DS, Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem 1981;114:193-7.
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