- Centrifuge 1.5 ml of a bacterial culture (grown overnight) for 1 min at maximal speed in a microcentrifuge (room temperature or 4°C).
- Resuspend the pellet by vortexing in 100 µl of ice-cold solution: 50 mM glucose, 25 mM Tris-HCl (pH 8.0), 10 mM EDTA (pH 8.0). Incubate for 5 min at room temperature.
- Add 200 µl of a freshly prepared solution of 0.2 M NaOH with 1% SDS. Mix the contents by inverting the tube rapidly several times. Do not vortex to avoid shearing of chromosomal DNA! Incubate the mixture on ice up to 5 minutes
- Add 150 µl of ice-cold 3 M potassium acetate (pH 4.8) and mix by inverting the tube rapidly several times. Incubate on ice for 3-5 min. A white precipitate will form.
- Centrifuge the suspension at maximum speed for 5 min at 4°C and transfer the supernatant to a fresh tube.
- Add 1 volume of isopropanol and vortex.
- Centrifuge the suspension at maximum speed for 20 min at 4°C. Then, wash the pellet with 300 µl of cold 70% ethanol.
- Air-dry the pellet and resuspend the DNA in 50 µl of water, nuclease-free.
- Add 2 µl of RNase A/T1 RNase Mix to the mixture and incubate at 37°C for 30 min.
- Then, remove enzymes from the DNA preparation using phenol/chloroform extraction (see protocol).
The prepared DNA is ready for sequencing and digestion with restriction enzymes.
The procedure yields 1-3 µg of plasmid DNA.Note
- Glucose may not be autoclaved, therefore purify the glucose stock solution by sterile filtration.
- 0.2 M NaOH with 1% SDS solution should be freshly prepared.
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Updated gegužės 09, 2007 17:25
