Team:Groningen/protocols/Genomic DNA extraction
From 2013.igem.org
Genomic DNA extraction
Materials:
- 1.5 ml microcentrifuge tubes
- 2 ml microcentrifuge tubes
- heatblock, 80°C
- stove, 65°C
- heatblock, 55°C
- isopropanol RT
- 70% ethanol RT
- BBA + lysozyme + RNAse (Birnboim solution A chromosomal DNA isolation + 5 mg/ml lysozyme + 3 µl RNAse (pinch of RNAse)
- Promega Nuclei Lysis Solution CAT# A7943
- Promega Precipitation Solution CAT# A7953
Reaction procedure:
- Add 2-4 ml of an overnight culture to a 1.5/2 ml microcentrifuge tube.
- Centrifuge at 14000g for 1 minute to pellet the cells. Remove the supernatant.
- Resuspend the cells thoroughly in 300 μl BBA + lysozyme + RNase.
- Incubate the sample at 55°C for 10 minutes.
- Add 500 μl of Nuclei Lysis Solution. The suspension should be clear.
- Incubate at 80°C for 5 minutes to lyse the cells; then cool to room temperature.
- Add 200 μl of Protein Precipitation Solution to the RNase-treated cell lysate. Vortex vigorously at high speed for 20 seconds to mix the Protein Precipitation Solution with the cell lysate. The suspension should be ‘milky’ colored.
- Incubate at ice for 10 minutes.
- Centrifuge at 14000g for 10 minutes.
- Transfer the supernatant containing the DNA to a clean 1.5/2 ml microcentrifuge tube containing 600/800 μl isopropanol. Fill it up to 1.5/2 ml with isopropanol.
- Gently mix by inversion until thread-like strands of DNA form a visible mass.
- Centrifuge at 14000g for 10 minutes.
- 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.
- Centrifuge at 14000g for 5 minutes. Carefully aspirate the ethanol.
- Drain the tube on clean absorbent paper and allow the pellet to air-dry for 10-15 minutes (stove at 45°C).
- Add 100 μl TE buffer or water (Elution Buffer from the Roche Purification Kit) to the tube and rehydrate the DNA by incubating at 65°C for 15 minutes. Periodically mix the solution by gently tapping the tube.
- Transfer the buffer + DNA to a 1.5 ml microcentrifuge tube. Store the DNA at 2-8°C.