You will be given a 1.5 ml microcentrifuge tube containing a pellet of Escherichia coli, strain BW30270. This strain is a K-12 or laboratory strain of E.coli. The pellets are made up of cells from 3 ml of overnight growth in a rich medium.
1. Add 600 μl of Nuclei Lysis Solution. Gently pipet up and down until the cells are resuspended.
2. Incubate at 80°C for 5 minutes to lyse the cells; then cool to room temperature.
3. Add 3μl of RNase Solution to the cell lysate. Invert the tube 2–5 times to mix.
4. Incubate at 37°C for 30 minutes. Cool the sample to room temperature.
5. Add 200 μl of Protein Precipitation Solution to the RNase-treated cell lysate.
6. Vortex vigorously at high speed for 20 seconds to mix the Protein Precipitation Solution with the cell lysate, be sure that the two solutions have completely mixed. Do not over-vortex or you risk shearing the chromosome.
7. Incubate the sample on ice for 5 minutes. Centrifuge at 13,000 rpm for 10 minutes.
8. Transfer the supernatant containing the DNA to a clean 1.5ml micro-centrifuge tube containing 600μl of room temperature isopropanol. Do not carry over any flecks of the precipitate.
9. Gently mix by inversion until the thread-like strands of DNA form a visible mass. If you do not see threads but a general whitish appears mix well and continue.
10. Centrifuge at 13,000 rpm for 5 minutes.
11. Carefully pour off the supernatant and drain the tube on clean absorbent paper. Add 600 μl of room temperature 70% ethanol and gently invert the tube several times to wash the DNA pellet.
12. Centrifuge at 13,000 rpm for 2 minutes. Carefully aspirate the ethanol from the tube with a pipet. Be careful not to suck up your pellet.
13. Drain the tube on kimwipes and allow the pellet to air-dry for 10–15 minutes.
14. Add 100μl of sterile water to the tube and rehydrate the DNA.
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